3&#39; Equatorial-Fluorine-Substituted Neuraminidase Inhibitor Compounds, Compositions and Methods for the Use Thereof as Anti-Virals

ABSTRACT

Equatorial 2,3-fluorinated glycosides compounds of formula (I) useful for the treatment or prophylaxis of viral infection, particularly viral influenza, the methods for their preparation, and their pharmaceutical compositions. The therapeutic effect is achieved via inhibition of viral neuraminidases.

TECHNICAL FIELD

This invention relates to therapeutics, their uses and methods for thetreatment or prophylaxis of viral infection. In particular the inventionrelates to compounds, compositions, therapies, and methods of treatmentfor viral infections such as influenza.

BACKGROUND

Infection and invasion by influenza viruses requires the intermediacy ofsialic acid residues on the surface of the host cell. The terms sialicacid and neuraminic acid are used interchangeably. Similarly, sialidaseand neuraminidase (NA) are used interchangeably. Initial attachment ofthe virus to the host cell occurs via the binding of the virus to thesesialic acids (charged, 9-carbon sugars) through the hemagglutininprotein of the virus. Once inside the cell the virus replicates bytaking advantage of the host cellular machinery. However, in order toremain optimally infective, the virus has evolved an NA that cuts offthe sialic acid from the host cell surface to assist the virus inescaping the host cell to infect other cells. Failure to cut off thesialic acid from the host cell surface, results in retention of virusthrough attachment to the host cell.

The GH33 family of neuraminidases contains all the sialidases except theviral enzymes (GH34 family). The GH33 and GH34 families are distinctstructurally and by sequence (See Cantarel B L. et al. (2009); andHenrissat B. and Davies G J (1997) for background on Familyclassifications). Previous work has demonstrated that 2,3-difluorosialicacids (DFSAs) are effective inhibitors of GH33 NAs and that GH33 NAsproceed through a covalent intermediate (see for example, Watts, A. etal. (2003); Amaya, M. F. et al. (2004); Watts, A. G. and Withers, S. G.(2004); Watts, A. G. et al. (2006); Newstead, S. et al. (2008); Damager,I. et al. (2008); and Buchini, S. et al. (2008)). The most probablemechanism for the GH34 sialidase (i.e. viral sialidases) reported in theliterature is one involving an ion-pair intermediate (von Itzstein M.(2007)).

A number of compounds are known to inhibit NAs. Some well known NAinhibitors are alkene-containing sialic acid analogues (for example:Laninamivir CAS #203120-17-6; Oseltamivir (Tamiflu) CAS #204255-11-8;and Zanamivir (Relenza) CAS #139110-80-8; see also U.S. Pat. No.5,360,817; and Ikeda et al. Bioorganic & Medicinal Chemistry (2006)14:7893-7897). Fluorinated sugar derivatives with (reactive) fluorideleaving groups have been shown to be inhibitors of a range of“retaining” glycosidases and function via formation of particularlystable glycosyl-enzyme intermediates (for example, Zechel and Withers((2000) Accounts of Chemical Research 33, 11-18) and Buchini et al.(2008)). These reagents are quite specific with respect to their targetenzymes, have been shown to be highly bio-available, and even capable ofcrossing the blood-brain barrier. Such inhibitors are mechanism-based intheir action, making the development of resistance by viruses difficult,whereby any mutations in the viral enzyme that reduce the inhibitionmust necessarily reduce the efficiency of the enzyme on the naturalsubstrate, sialic acid and therefore less likely to be tolerated. Theinitial design of oseltamivir and zanamivir was based upon the mimicryof the flattened transition state conformation of the sugar throughincorporation of an endocyclic alkene within a carbocycle (oseltamivir)or a pyranose ring (zanamivir) (M. von Itzstein et al., Nature 363, 418(1993)). Specificity for the influenza enzyme over other NAs, along withadditional affinity, was provided by incorporation of a guanidinium orammonium substituent at the position corresponding to C-4 of the naturalsubstrate to interact with a highly conserved anionic pocket at thatlocation in the active site. These broad spectrum influenza drugs areactive against NAs from group 1 and 2 influenza A strains as well asinfluenza B.

Despite being transition state analogue inhibitors, the emergence ofdrug-resistant strains has been reported, particularly against the morewidely used and structurally divergent drug oseltamivir. Mutations canbe both drug- and influenza subtype-specific. The most commonly seenmutation in viruses with the N1 subtype is H275Y which interferes withbinding of the isopentyl side chain of oseltamivir, but still permitsbinding of zanamivir and the natural substrate. Mutations most commonlydetected in clinical isolates with the N2 subtype include R292K (J. L.McKimm-Breschkin et al., J. Virol. 72, 2456 (1998); M. Tashiro et al.,Antivir. Ther. 14, 751 (2009); M. Kiso et al., Lancet 364, 759 (2004))and E119V (M. Tashiro et al., Antivir. Ther. 14, 751 (2009); M. Kiso etal., Lancet 364, 759 (2004)). Like the H275Y, the R292K precludes fullrotation of the E276 necessary to create the hydrophobic pocket thataccommodates the pentyl side-chain of oseltamivir (J. N. Varghese etal., Structure 6, 735 (1998)). In contrast, E119V confers oseltamivirspecific resistance due to altered interactions with the 4-amino group.E119A, D, G mutations seen in vitro (T. J. Blick et al., Virology 214,475 (1995); L. V. Gubareva et al., J. Virol. 71, 3385 (1997)) affectbinding of oseltamivir and/or zanamivir, demonstrating the significanceof the interactions of C-4 amino or guanidino group for high affinitybinding. Some of the recent mutations seen in pandemic H1N1 viruses,including I223R, confer reduced sensitivity to both inhibitors (A.Eshaghi et al., Emerg. Infect. Dis. 17, 1472 (2011); H. T. Nguyen etal., Clin. Infect. Dis. 51, 983 (2010); E. van der Vries et al., N.Engl. J. Med. 363, 1381 (2010)). The emergence of mutant strainssuggests that new neuraminidase inhibitors with increased propensity tomaintain potency against such mutant viral strains would be of interest.

SUMMARY

This invention is based in part on the fortuitous discovery thatcompounds having a 3′ equatorial fluorine (F), as described herein, havesurprisingly good neuraminidase modulatory properties. Specifically,compounds identified herein, show prolonged inhibition of neuraminidase,which may be useful for the treatment or prophylaxis of viral infection.In particular, compounds identified herein may be useful for thetreatment or prophylaxis of influenza. Moreover, the subjectcompositions exhibit a greater propensity to maintain potency againstparticular mutant viral strains as compared to correspondingstereoisomer compositions having a 3′axial F configuration and otherrelated compositions.

The compounds described herein may be used for in vivo or in vitroresearch uses (i.e. non-clinical) to investigate the mechanisms ofneuraminidase inhibition. Furthermore, these compounds may be usedindividually or as part of a kit for in vivo or in vitro research toinvestigate neuraminidase inhibition using recombinant proteins, viralstrains, cells maintained in culture, and/or animal models.Alternatively, the compounds described herein may be combined withcommercial packaging and/or instructions for use.

This invention is also based in part on the discovery that the compoundsdescribed herein, may also be used to modulate neuraminidase activityeither in vivo or in vitro for both research and therapeutic uses. Thecompounds may be used in an effective amount so that neuraminidaseactivity may be modulated. The neuraminidase may be viral. Theneuraminidase may be an influenza neuraminidase. In particular, thecompounds may be used to inhibit viral neuraminidase activity. Thecompounds modulatory activity may be used in either an in vivo or an invitro model for the study of viral infection. For example, influenzainfection. Furthermore, the compounds modulatory activity may be usedfor the treatment or prophylaxis of viral infection. The viral infectionmay be influenza.

In accordance with one embodiment, there are provided compounds having a3′ equatorial configuration of Formula I:

wherein T may be COOH or COOR¹, wherein R¹ may be a C₁₋₂₀ linear,branched or cyclic, saturated or unsaturated, unsubstituted alkyl group,Z may be F, or Cl; D may be F, or Cl; X may be NH₂, NHC(NH)NH₂, NHCH₃,NHCH₂CH₃, NHCH₂CH₂CH₃, NHCH₂CH₂CH₂CH₃, or NHC(CH₃)CH₃; Q may be OH, OMe,or OAc; E may be OH, or OAc; and A may be OH, or OAc.

In accordance with a further embodiment, there is provided a 3′equatorial compound, having the formula:

In accordance with a further embodiment, there is provided a 3′equatorial compound having the formula:

In accordance with a further embodiment, there are provided compounds asdescribed herein for modulating viral neuraminidase activity. The viralneuraminidase may be a GH34 neuraminidase. The modulating of viralneuraminidase activity may be for the treatment of influenza in ananimal. The animal may be a mammal. The animal may be a human.

In accordance with a further embodiment, there are provided compounds asdescribed herein for use in the preparation of a medicament formodulating viral neuraminidase activity. The viral neuraminidase may bea GH34 neuraminidase. The modulating of viral neuraminidase activity maybe for the treatment of influenza in an animal. The animal may be amammal. The animal may be a human.

In accordance with a further embodiment, there are provided compounds asdescribed herein for use in modulating viral neuraminidase activity. Theviral neuraminidase may be a GH34 neuraminidase. The modulating of viralneuraminidase activity may be for the treatment of influenza in ananimal. The animal may be a mammal. The animal may be a human.

In accordance with a further embodiment, there are providedpharmaceutical compositions which may include one or more compounds asdescribed herein, or pharmaceutically acceptable salts thereof, and apharmaceutically acceptable excipient. The pharmaceutical compositionsare useful for modulating viral neuraminidase activity. The viralneuraminidase may be a GH34 neuraminidase. The modulating of viralneuraminidase activity may be for the treatment of influenza in ananimal. The animal may be a mammal. The animal may be a human.

In accordance with a further embodiment, there are providedpharmaceutically acceptable salts of compounds described herein formodulating viral neuraminidase activity. The viral neuraminidase may bea GH34 neuraminidase. The modulating of viral neuraminidase activity maybe for the treatment of influenza in an animal. The animal may be amammal. The animal may be a human.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt(s) thereof, or apharmaceutical composition comprising the same. In a further embodiment,there is provided a method of inhibiting viral neuraminidase activity.The viral neuraminidase may be a GH34 neuraminidase. The modulating ofviral neuraminidase activity may be for the treatment of influenza in ananimal. The animal may be a mammal. The animal may be a human.

In accordance with a further embodiment, there is provided a commercialpackage which may contain one or more compounds described herein or apharmaceutically acceptable salt thereof or a pharmaceutical compositionthereof. The commercial package may optionally contain instructions forthe use of the compounds or pharmaceutically acceptable salt(s) thereofor pharmaceutical composition comprising the same in the treatment ofinfluenza.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity comprising the use of a 3′equatorial compound described herein or a pharmaceutically acceptablesalt thereof, or a pharmaceutical composition comprising the same,wherein the compound (or pharmaceutically acceptable salt thereof) hasat least about 2 fold better maintenance of potency as against a mutantvirus as compared to its corresponding stereoisomer having a 3′ axialconfiguration. In one embodiment, the method involves the use of morethan one 3′ equatorial compound provided herein, wherein at least one ofthe compounds has at least about 2 fold better maintenance of potency asagainst a mutant virus as compared to its corresponding steroeooisomerhaving a 3′ axial configuration.

Furthermore, 3′ equatorial compounds may be used in combination withnon-formula 1 compounds (for example, 3′ axial compounds) forcombination therapy.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about 2fold better maintenance of potency as against a resistant virus ascompared to the corresponding steroeioosomer having a 3′ axialconfiguration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about 2fold better maintenance of potency as against a virus as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about 2fold increase in specificity for a viral neuraminidase target (i.e.ki/Ki value) as compared to the corresponding steroeioosomer having a 3′axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about 3fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about 4fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about 5fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about10 fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about15 fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about19 fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about20 fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about25 fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a method ofmodulating viral neuraminidase activity with one or more compoundsdescribed herein or a pharmaceutically acceptable salt thereof, whereinthe composition having 3′ equatorial configuration, has at least about30 fold increase in specificity (i.e. ki/Ki value) as compared to thecorresponding steroeioosomer having a 3′ axial configuration.

In accordance with a further embodiment, there is provided a commercialpackage comprising one or more compounds or composition having 3′equatorial configuration described herein or a pharmaceuticallyacceptable salt thereof as described herein and instructions for the usein modulating viral neuraminidase activity.

T may be COOEt; Z may be F; D may be F; X may be NH₂ or NHC(NH)NH₂; Qmay be OH; E may be OH; and A may be OH.

T may be COOH; Z may be F; D may be F; X may be NH₂ or NHC(NH)NH₂; Q maybe OH; E may be OH; and A may be OH.

T may be COOH; Z may be F; D may be F; X may be NH₂; Q may be OH; E maybe OH; and A may be OH.

T may be COOH; Z may be F; D may be F; X may be NHC(NH)NH₂; Q may be OH;E may be OH; and A may be OH.

R¹ may be C₁₋₁₉. R¹ may be C₁₋₁₈. R¹ may be C₁₋₁₇. R¹ may be C₁₋₁₆. R¹may be C₁₋₁₅. R¹ may be C₁₋₁₄. R¹ may be C₁₋₁₃. R¹ may be C₁₋₁₂. R¹ maybe C₁₋₁₁. R¹ may be C₁₋₁₀. R¹ may be C₁₋₉. R¹ may be C₁₋₈. R¹ may beC₁₋₇. R¹ may be C₁₋₆. R¹ may be C₁₋₅. R¹ may be C₁₋₄. R¹ may be C₁₋₃. R¹may be C₁₋₂. R¹ may be C₁. R¹ may be C₂₋₁₀. R¹ may be C₂₋₃. R¹ may beC₂₋₄. R¹ may be C₂₋₅. R¹ may be C₂₋₆. R¹ may be C₂₋₇. R¹ may be C₂₋₈. R¹may be C₂₋₉. R¹ may be C₂₋₁₀. R¹ may be C₂₋₁₁. R¹ may be C₂₋₁₂. R¹ maybe C₂₋₁₃. R¹ may be C₂₋₁₄. R¹ may be C₂₋₁₅. R¹ may be C₂₋₁₆. R¹ may beC₂₋₁₇. R¹ may be C₂₋₁₈. R¹ may be C₂₋₁₉. R¹ may be C₂₋₂₀. R¹ may beC₃₋₁₀. R¹ may be C₄₋₁₀. R¹ may be C₅₋₁₀. R¹ may be C₆₋₁₀. R¹ may beC₇₋₁₀. R¹ may be C₈₋₁₀.

R¹ may be C₉₋₁₀. Alternatively, R¹ may be optionally substituted. Theoptional substituent may be selected from one or more of the groupincluding of: oxo, OH, F, Cl, Br, I, NH₂, CN, SH, SO₃H and NO₂, and zeroto ten backbone carbons of the optionally substituted alkyl group may beoptionally and independently substituted with O, N or S. T may be COOH.T may be COOEt. T may be COOPr. T may be COOBu. T may be COOMe. R¹ maybe linear. R¹ may be branched. R¹ may be cyclic. R¹ may be saturated. R¹may be unsaturated.

Z may be F or Cl. D may be F or Cl. Z may be F. D may be F. Z may be Cl.D may be Cl. Z may be F and D may be F or Cl. Z may be Cl and D may be For Cl. Z may be Cl and D may be Cl. Z may be F and D may be Cl. Z may beF and D may be F. Z may be F and D may be F or Cl. Z may be F or Cl andD may be F. Z may be F or Cl and D may be Cl.

X may be NH₂, NHC(NH)NH₂, NHCH₃, NHCH₂CH₃, NHCH₂CH₂CH₃, NHCH₂CH₂CH₂CH₃,or NHC(CH₃)CH₃. X may be NH₂. X may be NHC(NH)NH₂. X may be NHCH₃. X maybe NHCH₂CH₃. X may be NHCH₂CH₂CH₃. X may be NHCH₂CH₂CH₂CH₃. X may beNHC(CH₃)CH₃. X may be NH₂, or NHC(NH)NH₂. X may be NH₂, NHC(NH)NH₂, orNHCH₃. X may be NH₂, NHC(NH)NH₂, or NHC(CH₃)CH₃.

Q may be OH, OMe, or OAc. Q may be OH, or OAc. Q may be OAc. Q may beOH. Q may be OMe. Q may be OH, or OMe, or OAc. Q may be OMe, or OAc.

E may be OH, OMe, or OAc. E may be OH, or OAc. E may be OAc. E may beOH. E may be OMe. E may be OH, or OMe, or OAc. E may be OMe, or OAc.

A may be OH, OMe, or OAc. A may be OH, or OAc. A may be OAc. A may beOH. A may be OMe. A may be OH, or OMe, or OAc. A may be OMe, or OAc.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in greater detail with reference tothe accompanying diagrams.

FIG. 1 is a schematic depiction of the mechanism of action of DFSAs andthe X-ray structure of inhibited enzyme, including the following: a) themechanism of action of the 2,3-difluorosialic acids (DFSAs); b) theX-ray crystallographic structure of the active site of an NA trapped asits 3-fluoro(eq)-4-guanidino-sialyl-enzyme intermediate (contacts ≦3 Åare shown with dashed lines; and c) a diagram of the interactions (≦3 Å;red dashed lines) with the sugar in the covalently inhibited NA.

FIG. 2 is a graphical representation of (a) the inactivation ofinfluenza NA N9 as a function of time upon incubation with difluoroKDNand (b) the spontaneous time-dependent reactivation of influenza NA N9activity after removal of excess Difluoro KDN fromdifluoroKDN-inactivated NA-N9.

FIG. 3 is a schematic depiction of the LC/MS daughter-ion fragmentationpattern of digested influenza NA N9 inactivated with Difluoro KDN, whichconfirms the covalent attachment of the inhibitor to Tyr406.

FIG. 4 is a graphical representation of the efficacy of FaxGuDFSA,FeqGuDFSA and zanamivir in treating H3N2 influenza infection in theBalb/c mouse. The top graph of FIG. 4 a shows body weight over the 17day observation period. Animals that lost 20% of initial weight wererecorded as non-survivors, as indicated in the survival plot (inset). Ina parallel set of animals (bottom graph of FIG. 4 a), viral RNA loadswere measured over 7 days by qPCR. In this group, all untreated animalssurvived only 3 days. FIG. 4 b shows the dose dependent efficacy ofFeqGuDFSA (1-10 mg/kg/d) and zanamivir. FeqGuDFSA was partly (20%)effective at protecting animals at 3 mg/kg/d ((*) Mantel Cox p=0.03),whereas a 10 mg/kg/d dose was 100% effective (dotted line, (***) MantelCox p<0.001).

DETAILED DESCRIPTION

As used herein, the phrase “C_(x-y) alkyl” or “C_(x)-C_(y) alkyl” isused as it is normally understood to a person of skill in the art andoften refers to a chemical entity that has a carbon skeleton or maincarbon chain comprising a number from x to y (with all individualintegers within the range included, including integers x and y) ofcarbon atoms. For example a “C₁₋₁₀ alkyl” is a chemical entity that has1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atom(s) in its carbon skeleton ormain chain. Alternatively, for example a “C₁₋₂₀ alkyl” is a chemicalentity that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 carbon atom(s) in its carbon skeleton or main chain.

As used herein, the term “branched” is used as it is normally understoodto a person of skill in the art and often refers to a chemical entitythat comprises a skeleton or main chain that splits off into more thanone contiguous chain. The portions of the skeleton or main chain thatsplit off in more than one direction may be linear, cyclic or anycombination thereof. Non-limiting examples of a branched alkyl aretert-butyl and isopropyl.

As used herein, the term “unbranched” is used as it is normallyunderstood to a person of skill in the art and often refers to achemical entity that comprises a skeleton or main chain that does notsplit off into more that one contiguous chain. Non-limiting examples ofunbranched alkyls are methyl, ethyl, n-propyl, and n-butyl.

As used herein, the term “substituted” is used as it is normallyunderstood to a person of skill in the art and often refers to achemical entity that has one chemical group replaced with a differentchemical group that contains one or more heteroatoms. Unless otherwisespecified, a substituted alkyl is an alkyl in which one or more hydrogenatom(s) is/are replaced with one or more atom(s) that is/are nothydrogen(s). For example, chloromethyl is a non-limiting example of asubstituted alkyl, more particularly an example of a substituted methyl.Aminoethyl is another non-limiting example of a substituted alkyl, moreparticularly it is a substituted ethyl. The functional groups describedherein may be substituted with, for example, and without limitation, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents.

As used herein, the term “unsubstituted” is used as it is normallyunderstood to a person of skill in the art and often refers to achemical entity that is a hydrocarbon and/or does not contain aheteroatom. Non-limiting examples of unsubstituted alkyls includemethyl, ethyl, tert-butyl, and pentyl.

As used herein, the term “saturated” when referring to a chemical entityis used as it is normally understood to a person of skill in the art andoften refers to a chemical entity that comprises only single bonds.Non-limiting examples of saturated chemical entities include ethane,tert-butyl, and N⁺H₃.

As used herein the term “halogenated” is used as it would normally beunderstood to a person of skill in the art and refers to a moiety orchemical entity in which a hydrogen atom is replaced with a halogen atomsuch as chlorine, fluorine, iodine or bromine. For example, afluorinated side chain refers to a side chain wherein one or morehydrogen atoms is replaced with one or more fluorine atoms.

Non-limiting examples of saturated C₁-C₁₀ alkyl may include methyl,ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl,t-butyl, n-pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl, i-hexyl,1,2-dimethylpropyl, 2-ethylpropyl, 1-methyl-2-ethylpropyl,1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1,2-triethylpropyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, 1,3-dimethylbutyl,2-methylpentyl, 3-methylpentyl, sec-hexyl, t-hexyl, n-heptyl, i-heptyl,sec-heptyl, t-heptyl, n-octyl, i-octyl, sec-octyl, t-octyl, n-nonyl,i-nonyl, sec-nonyl, t-nonyl, n-decyl, i-decyl, sec-decyl and t-decyl.Non-limiting examples of C₂-C₁₀ alkenyl may include vinyl, allyl,isopropenyl, 1-propene-2-yl, 1-butene-1-yl, 1-butene-2-yl,1-butene-3-yl, 2-butene-1-yl, 2-butene-2-yl, octenyl and decenyl.Non-limiting examples of C₂-C₁₀ alkynyl may include ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl.Saturated C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl or C₂-C₁₀ alkynyl may be, forexample, and without limitation, interrupted by one or more heteroatomswhich are independently nitrogen, sulfur or oxygen.

The embodiments involving the formulae as described herein include allpossible stereochemical alternatives except where expressly excluded,including those illustrated or described herein.

In some embodiments, the compounds as described herein or acceptablesalts thereof above may be used for systemic treatment or prophylaxis ofa viral infection. In some embodiments, the compounds as describedherein or acceptable salts thereof above may be used in the preparationof a medicament or a composition for systemic treatment or prophylaxisof a viral infection. In some embodiments, methods of systemicallytreating any of the infections described herein are also provided. Someembodiments, make use of compositions comprising a compound describedherein and a pharmaceutically acceptable excipient or carrier. In someembodiments, the viral infection is caused, at least in part, by aninfluenza virus. Methods of treating any of the indications describedherein are also provided. Such methods may include administering acompound as described herein or a composition comprising a compound asdescribed herein, or an effective amount of a compound as describedherein or composition comprising a compound as described herein to asubject in need thereof.

Compounds as described herein may be in the free form or in the form ofa salt thereof. In some embodiments, compounds as described herein maybe in the form of a pharmaceutically acceptable salt, which are known inthe art (Berge et al., J. Pharm. Sci. (1977) 66:1). Pharmaceuticallyacceptable salt as used herein includes, for example, salts that havethe desired pharmacological activity of the parent compound (salts whichretain the biological effectiveness and/or properties of the parentcompound and which are not biologically and/or otherwise undesirable).Compounds as described herein having one or more functional groupscapable of forming a salt may be, for example, formed as apharmaceutically acceptable salt. Compounds containing one or more basicfunctional groups may be capable of forming a pharmaceuticallyacceptable salt with, for example, a pharmaceutically acceptable organicor inorganic acid. Pharmaceutically acceptable salts may be derivedfrom, for example, and without limitation, acetic acid, adipic acid,alginic acid, aspartic acid, ascorbic acid, benzoic acid,benzenesulfonic acid, butyric acid, cinnamic acid, citric acid,camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid,diethylacetic acid, digluconic acid, dodecylsulfonic acid,ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid,gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid,heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid,hydriodic acid, 2-hydroxyethanesulfonic acid, isonicotinic acid, lacticacid, malic acid, maleic acid, malonic acid, mandelic acid,methanesulfonic acid, 2-napthalenesulfonic acid, naphthalenedisulphonicacid, p-toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid,pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid,picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid,salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaricacid, thiocyanic acid or undecanoic acid. Compounds containing one ormore acidic functional groups may be capable of forming pharmaceuticallyacceptable salts with a pharmaceutically acceptable base, for example,and without limitation, inorganic bases based on alkaline metals oralkaline earth metals or organic bases such as primary amine compounds,secondary amine compounds, tertiary amine compounds, quaternary aminecompounds, substituted amines, naturally occurring substituted amines,cyclic amines or basic ion-exchange resins. Pharmaceutically acceptablesalts may be derived from, for example, and without limitation, ahydroxide, carbonate, or bicarbonate of a pharmaceutically acceptablemetal cation such as ammonium, sodium, potassium, lithium, calcium,magnesium, iron, zinc, copper, manganese or aluminum, ammonia,benzathine, meglumine, methylamine, dimethylamine, trimethylamine,ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine,tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine,glucamine, methylglucamine, theobromine, purines, piperazine,piperidine, procaine, N-ethylpiperidine, theobromine,tetramethylammonium compounds, tetraethylammonium compounds, pyridine,N,N-dimethylaniline, N-methylpiperidine, morpholine, N-methylmorpholine,N-ethylmorpholine, dicyclohexylamine, dibenzylamine,N,N-dibenzylphenethylamine, 1-ephenamine, N,N′-dibenzylethylenediamineor polyamine resins. In some embodiments, compounds as described hereinmay contain both acidic and basic groups and may be in the form of innersalts or zwitterions, for example, and without limitation, betaines.Salts as described herein may be prepared by conventional processesknown to a person skilled in the art, for example, and withoutlimitation, by reacting the free form with an organic acid, an inorganicacid, an organic base or an inorganic base, or by anion exchange orcation exchange from other salts. Those skilled in the art willappreciate that preparation of salts may occur in situ during isolationand/or purification of the compounds or preparation of salts may occurby separately reacting an isolated and/or purified compound.

In some embodiments, compounds and all different forms thereof (e.g.free forms, salts, polymorphs, isomeric forms) as described herein maybe in the solvent addition form, for example, solvates. Solvates containeither stoichiometric or non-stoichiometric amounts of a solvent inphysical association with the compound or salt thereof. The solvent maybe, for example, and without limitation, a pharmaceutically acceptablesolvent. For example, hydrates are formed when the solvent is water oralcoholates are formed when the solvent is an alcohol.

In some embodiments, compounds and all different forms thereof (e.g.free forms, salts, solvates, isomeric forms) as described herein mayinclude crystalline and/or amorphous forms, for example, polymorphs,pseudopolymorphs, conformational polymorphs, amorphous forms, or acombination thereof. Polymorphs include different crystal packingarrangements of the same elemental composition of a compound. Polymorphsusually have different X-ray diffraction patterns, infrared spectra,melting points, density, hardness, crystal shape, optical and electricalproperties, stability and/or solubility. Those skilled in the art willappreciate that various factors including recrystallization solvent,rate of crystallization and storage temperature may cause a singlecrystal form to dominate.

In some embodiments, compounds and all different forms thereof (e.g.free forms, salts, solvates, polymorphs) as described herein may includeisomers such as geometrical isomers, optical isomers based on asymmetriccarbon, stereoisomers, tautomers, individual enantiomers, individualdiastereomers, racemates, diastereomeric mixtures and combinationsthereof, and are not limited by the description of the formulaillustrated for the sake of convenience unless specifically excluded.

In some embodiments, pharmaceutical compositions in accordance with thisinvention may comprise a salt of such a compound, preferably apharmaceutically or physiologically acceptable salt. Pharmaceuticalpreparations will typically comprise one or more carriers, excipients ordiluents acceptable for the mode of administration of the preparation,be it by injection, inhalation, oral, topical administration, lavage, orother modes suitable for the selected treatment. Suitable carriers,excipients or diluents include those known in the art for use in suchmodes of administration.

Suitable pharmaceutical compositions may be formulated by means known inthe art and their mode of administration and dose determined by theskilled practitioner. For parenteral administration, a compound may bedissolved in sterile water or saline or a pharmaceutically acceptablevehicle used for administration of non-water soluble compounds such asthose used for vitamin K. For enteral administration, the compound maybe administered in a tablet, capsule or dissolved in liquid form. Thetablet or capsule may be enteric coated, or in a formulation forsustained release. Many suitable formulations are known, including,polymeric or protein microparticles encapsulating a compound to bereleased, ointments, pastes, gels, hydrogels, or solutions which can beused topically or locally to administer a compound. A sustained releasepatch or implant may be employed to provide release over a prolongedperiod of time. Many techniques known to one of skill in the art aredescribed in Remington: the Science & Practice of Pharmacy by AlfonsoGennaro, 20^(th) ed., Lippencott Williams & Wilkins, (2000).Formulations for parenteral administration may, for example, containexcipients, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, or hydrogenated naphthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for modulatory compounds include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel. The formulations may be specifically prepared for intranasaldelivery. For example, nasal inhalation.

Compounds or pharmaceutical compositions in accordance with thisinvention or for use in this invention may be administered by means of amedical device or appliance such as an implant, graft, prosthesis,stent, etc. Also, implants may be devised which are intended to containand release such compounds or compositions. An example would be animplant made of a polymeric material adapted to release the compoundover a period of time.

An “effective amount” of a pharmaceutical composition as describedherein includes a therapeutically effective amount or a prophylacticallyeffective amount. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result, such as reduced viral load,increased life span or increased life expectancy. A therapeuticallyeffective amount of a compound may vary according to factors such as thedisease state, age, sex, and weight of the subject, and the ability ofthe compound to elicit a desired response in the subject. Dosageregimens may be adjusted to provide the optimum therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the compound are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result, such as lesssevere infection or delayed or no onset, increased life span, increasedlife expectancy or prevention of the progression of infection.Typically, a prophylactic dose is used in subjects prior to or at anearlier stage of disease, so that a prophylactically effective amountmay be less than a therapeutically effective amount.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. For any particular subject, specific dosageregimens may be adjusted over time according to the individual need andthe professional judgment of the person administering or supervising theadministration of the compositions. Dosage ranges set forth herein areexemplary only and do not limit the dosage ranges that may be selectedby medical practitioners. The amount of active compound(s) in thecomposition may vary according to factors such as the disease state,age, sex, and weight of the subject. Dosage regimens may be adjusted toprovide the optimum therapeutic response. For example, a single bolusmay be administered, several divided doses may be administered over timeor the dose may be proportionally reduced or increased as indicated bythe exigencies of the therapeutic situation. It may be advantageous toformulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage.

In some embodiments, compounds and all different forms thereof asdescribed herein may be used, for example, and without limitation, incombination with other treatment methods.

In general, compounds described herein should be used without causingsubstantial toxicity. Toxicity of the compounds of the invention can bedetermined using standard techniques, for example, by testing in cellcultures or experimental animals and determining the therapeutic index(evaluate the animals for acute and chronic signs of injury afteradministration of the drug and determining the maximum tolerated dose(MTD) as for example the dose at which no adverse effects are observedor at which only tolerable effects are observed, and then be compared tothe effective dose and ratio of MTD to effective dose becomes thetherapeutic index). In some circumstances, however, such as in severedisease conditions, it may be necessary to administer substantialexcesses of the compositions. Some compounds described herein may betoxic at some concentrations. Titration studies may be used to determinetoxic and non-toxic concentrations. Toxicity may be evaluated byexamining a particular compound's or composition's specificity acrosscell lines. Animal studies may be used to provide an indication if thecompound has any effects on other tissues.

Compounds as described herein may be administered to a subject. As usedherein, a “subject” may be a human, non-human primate, rat, mouse, cow,horse, pig, sheep, goat, dog, cat, etc. The subject may have, be at riskfor having, be suspected of having or being at risk for having aninfection, such as a viral infection. In particular, the infection maybe facilitated by or mediated by or assisted by a neuraminidase.Diagnostic methods for viral infection, such as influenza and theclinical delineation of viral infection, such as influenza are known tothose of ordinary skill in the art.

TABLE 1A Comparative Test of Compounds for Neuraminidase ModulatoryActivity.

TABLE 1A 2,3-Fluorinated Glycosides having both a 3 equatorial and 3axial Fluorines (DFSA and derivatives thereof) Compound Structure DFSA

FaxAmDFSA (Compound 4NH₂3Fax)

4-G 3 F equatorial diF SA (4-G 3Feq diFSA) (FeqGuDFSA) (Compound 4G3Feq)

 

4-NH₂ 3 F equatorial diF SA (2F3F4NH₂) (FeqAmDFSA) (Compound 4NH₂3Feq)

4-G diF SA (FaxGuDFSA) (Compound 4G3Fax)

TABLE 1B Known Anti-Viral Compounds for Comparative Activity Testing.

TABLE 1B Known Anti-viral Compounds Compound Structure Zanamivir (Cass #139110-80-8)

Peramivir (Cass # 229614-55-5)

Oseltamivir (Cass # 196618-13-0)

TABLE 2 New Compounds Having Surprisingly Good Neuraminidase ModulatoryProperties.

TABLE 2 Equatorial 2,3-Fluorinated Glycosides with NeuraminidaseModulatory Activity Compound Structure FeqGuDFSA

 

FeqAmDFSA

 

 

Compounds described herein may also be used in assays and for researchpurposes.

Compounds for use in the present methods may be synthesized using themethods described herein.

Various alternative embodiments and examples are described herein. Theseembodiments and examples are illustrative and should not be construed aslimiting the scope of the invention.

General Methodologies Synthesis

All chemicals were of analytical grade purchased from the Sigma-Aldrichcompany, unless otherwise stated. All solvents were BOC standard gradeand distilled before use. Dichloromethane was distilled from calciumhydride. Methanol was distilled from magnesium. N,N-dimethylformamideand DIPEA were dried and stored over 4 Å molecular sieves. Analyticalthin-layer chromatography (TLC) was performed on aluminum-backed sheetsof Silica Gel 60F₂₅₄ (E. Merck) of thickness 0.2 mm. The plates werevisualized using UV light (254 nm) and/or by exposure to 10% ammoniummolybdate (2 M in H₂SO₄) followed by charring. Flash columnchromatography was carried out using Merck Kieselgel 60 (230-400 mesh).Proton and carbon NMR spectra were recorded on Bruker Avance 400 inv,400 dir and 300 Fourier Transform spectrometers fitted with 5 mm BBI-Zprobes. Fluorine NMR spectra were recorded on a Bruker Avance 300 fittedwith a 5 mm QNP probe. All spectra were recorded using an internaldeuterium lock and are referenced internally using the residual solventpeak. Carbon and proton chemical shifts are quoted in parts per million(ppm) downfield of tetramethylsilane, fluorine chemical shifts arequoted downfield of trifluoroacetic acid. Coupling constants (J) aregiven in Hertz (Hz) and are quoted to the nearest 0.5 Hz. Carbon NMRspectra were performed with broadband proton decoupling and wererecorded with DEPT. ¹H-NMR experiments performed in D₂O solvent wererecorded with a water suppression protocol. Mass spectra were recordedon a Waters/Micromass LCT using electrospray ionization (ESI) andrecorded using the Time-Of-Flight (TOF) method using methanol assolvent.

General methodologies for chemical preparation of 3′ equatorialcompounds of Formula I are described in the following non-limitingexemplary schemes. Furthermore, additional modifications to the belowexemplary schemes and alternative syntheses as described inPCT/CA2010/001063.

Compounds of Formula I having modifications at Cl, may be prepared bythe chemical methodologies described in the following non-limitingexemplary scheme.

It will be appreciated by a person of skill in the art, that variationsin the alkyl chain length may be achieved by substituting 1-octanol(C8—having 8 carbons) for an alternative alcohol. For example, 1-octanolin the above scheme may be substituted for an alternative primaryalcohol, which may, for example, be selected from one or more of thefollowing: Propan-1-ol (C3); Butanol (C4); 1-Pentanol (C5); 1-Hexanol(C6); 1-Heptanol (C7); 1-Nonanol (C9); 1-Decanol (C10); Undecanol (C11);Dodecanol (C12); 1-Tetradecanol (C14); Cetyl alcohol (C16); Stearylalcohol (C18); and Arachidyl alcohol (C20). Similarly, it will beappreciated that an alternative substrate for this reaction may bechosen. For example, instead of the 4NH₂ (compound 7) the 4Gu compound(compound 5), or etc. may be substituted.

Alternatively, compounds of Formula I having modifications at C1, may beprepared by the chemical methodologies described in the followingnon-limiting exemplary scheme. For example, the below exemplary schemeadds an ethyl group at C1 (R¹). It will be appreciated by a person ofskill in the art, that variations in the salt produced may be achievedby substituting an alternative acid and that the length of the alkylgroup at C1 may be adjusted by substituting an alternative alcohol asset out above.

Syntheses and Characterizations

Methyl5-acetamido-7,8,9-tri-O-acetyl-4-azido-4,5-dideoxy-3β-fluoro-D-erythro-L-glucononulo-pyranosonate(2) “Methyl5-acetamido-7,8,9-tri-O-acetyl-4-azido-3,4,5-trideoxy-3β-fluoro-D-erythro-L-glucononulopyranosonate”:A suspension of 1 (11.1 g, 24.3 mmol), nitromethane (95 mL), water (16mL) and Selectfluor (34.5 g, 97.5 mmol, 4 equiv.) was stirred for 7 daysat room temperature. (The reaction may be monitored for completion by UVon TLC, because only the starting material is detected under short UV.The reaction is considered complete upon disappearance of the UV activecompound.). The reaction was quenched with saturated NaHCO₃ (100 mL),extracted with EtOAc (4×200 mL). The organic phase was washed withsaturated NaHCO₃ (300 mL) and brine (300 mL), dried over MgSO₄. Afterevaporation, the resulting residue was purified by flash columnchromatography (CHCl₃/Acetone/EtOAc=5/1/1) to give the desired compound2 as a white solid (2.14 g, 18%). ¹H NMR (400 MHz, CDCl₃): δ 5.73 (d,1H, J 9.2 Hz, NHAc), 5.30 (dd, 1H, J_(7,8) 6.7 Hz, H-7), 5.22 (m, 1H,H-8), 4.74 (dd, 1H, J_(3,4) 9.6 Hz, J_(H3,F3) 49.0 Hz, H-3), 4.66 (s,1H, OH), 4.40 (dd, 1H, J_(6,7) 1.8 Hz, J_(5,6) 10.5 Hz, H-6), 4.37 (dd,1H, J_(8,9a) 2.1 Hz, H-9a), 4.20 (m, 1H, H-4), 4.04 (dd, 1H, J_(8,9b)6.3 Hz, J_(9a,9b) 12.4 Hz, H-9b), 3.96 (s, 3H, OCH₃), 3.77 (m, 1H, H-5),2.15 (s, 3H, CH₃CO), 2.11 (s, 3H, CH₃CO), 2.04 (s, 3H, CH₃CO), 2.03 (s,3H, CH₃CO). ¹³C NMR (75 MHz, CDCl₃): δ 170.9, 170.7, 170.6 (2C), 167.8,93.3 (d, J_(C2,F3) 21.8 Hz, C-2), 89.2 (d, J_(C3,F3) 193.8 Hz, C-3),70.5, 69.6, 67.7, 62.6, 62.0 (d, J_(C4,F3) 17.2 Hz, C-4), 54.55, 49.9(d, J_(C5,F3) 6.0 Hz, C-5), 23.6, 21.2, 21.0, 20.9. ¹⁹F NMR (282 MHz,CDCl₃): δ −195.46 (s, F-2 eq). ESI-MS: 515.3 [(M+Na)⁺].

Methyl5-acetamido-7,8,9-tri-O-acetyl-4-azido-2,4,5-trideoxy-2α,3β-difluoro-α-D-erythro-L-gluco-nonulopyranosonate(3) “Methyl5-acetamido-7,8,9-tri-O-acetyl-4-azido-3,4,5-trideoxy-2α,3β-difluoro-α-D-erythro-L-glucononulopyranosonate”:To a suspension of 2 (0.62 g, 1.3 mmol) in dry DCM (18 mL) was addeddropwise DAST (0.18 mL, 1.4 mmol, 1.1 equiv) with stirring under N₂ at−40° C. After addition, the reaction mixture was stirred for 0.5 h at−40° C., and then gradually warmed up to −10° C. The reaction wasquenched with saturated NaHCO₃, diluted with DCM (50 mL) and washed withbrine (30 mL). The water phase was extracted again with EtOAc (2×50 mL)and washed with brine (50 mL). The combined organic phase was dried overMgSO₄. After evaporation, the resulting residue was purified by flashcolumn chromatography (DCM/Acetone=8/1) to give product 3 as a whitesolid (0.566 g, 91%). ¹H NMR (400 MHz, CDCl₃): δ 5.59 (d, 1H, J 9.0 Hz,NHAc), 5.32 (m, 1H, H-8), 5.24 (dt, 1H, J_(7,8) 8.5 Hz, H-7), 4.70 (dd,1H, J_(5,6) 10.7 Hz, J_(6,7) 1.6 Hz, H-6), 4.66 (ddd, 1H, J_(4,5) 10.7Hz, J_(H4,F3) 20.2 Hz, H-4), 4.47 (ddd, 1H, J_(3,4) 9.3 Hz, J_(H3,F3)48.6 Hz, J_(H3,F2) 14.5 Hz, H-3), 4.25 (dd, 1H, J_(8,9a) 2.6 Hz, H-9a),4.13 (dd, 1H, J_(8,9b) 5.2 Hz, J_(9a,9b) 12.5 Hz, H-9b), 3.91 (s, 3H,OCH₃), 3.62 (m, 1H, H-5), 2.14 (s, 3H, CH₃CO), 2.08 (s, 3H, CH₃CO), 2.05(s, 3H, CH₃CO), 2.04 (s, 3H, CH₃CO). ¹³C NMR (75 MHz, CDCl₃): δ 171.0,170.7, 170.5, 169.7, 165.3 (d, J_(C2,F2) 32.8 Hz, C-1), 105.5 (dd,J_(C2,F2) 229.1 Hz, J_(C2,F3) 27.2 Hz, C-2), 92.0 (dd, J_(C3,F3) 192.2Hz, J_(C3,F2) 29.0 Hz, C-3), 72.9, 68.9, 67.0, 62.2, 61.8 (dd, J_(C4,F3)18.1 Hz, J_(C4,F2) 8.4 Hz, C-4), 53.7, 49.2 (d, J_(C5,F3) 6.9 Hz, C-5),23.5, 21.0 (2C), 20.9. ¹⁹F NMR (282 MHz, CDCl₃): δ −119.4 (d, J_(F2,F3)12.7 Hz, F-2 eq), −197.5 (d, F-3 eq). ESI-MS: 517.2 [(M+Na)⁺].

Methyl5-acetamido-7,8,9-tri-O-acetyl-4-[(N′,N″-di-tert-butoxycarbonyl)guanidine]-2,4,5-trideoxy-2α,3β-difluoro-α-D-erythro-L-gluco-nonulpyranosonate(4) “Methyl5-acetamido-7,8,9-tri-O-acetyl-4-[(N′,N″-di-tert-butoxycarbonyl)guanidine]-3,4,5-trideoxy-2α,3β-difluoro-α-D-erythro-L-glucononulpyranosonate”:A mixture of 3 (260 mg, 0.53 mmol), EtOAc (10 mL), Pd/C (10%, 60 mg),N,N′-di-Boc-N″-trifluoromethanesulfonylguanidine (350 mg, 0.9 mmol, 1.7equiv) and DIPEA (0.2 mL) was placed under vacuum and then filled withhydrogen three times, and the mixture was stirred under a H₂ atmospherefor 24 h at room temperature. The reaction mixture was filtered througha short pad of Celite and washed with EtOAc. After evaporation, theresulting residue was purified by flash column chromatography(DCM/Acetone=15/1) to give the product 4 as a white solid (0.311 g,83%). ¹H NMR (400 MHz, CDCl₃): δ 11.37 (s, 1H, NHBoc), 8.67 (d, 1H, J7.8 Hz, NHGuanidine), 6.45 (d, 1H, J 9.0 Hz, NHAc), 5.32 (m, 1H, H-8),5.25 (brd, 1H, J_(7,8) 7.7 Hz, H-7), 4.90 (m, 1H, H-4), 4.71 (ddd, 1H,J_(3,4) 8.8 Hz, J_(H3,F3) 48.5 Hz, J_(H3,F2) 12.5 Hz, H-3), 4.50 (brd,1H, H-6), 4.32 (dd, 1H, J_(8,9a) 2.6 Hz, H-9a), 4.28 (m, 1H, H-5), 4.07(dd, 1H, J_(8,9b) 6.2 Hz, J_(9a,9b) 12.4 Hz, H-9b), 3.90 (s, 3H, OCH₃),2.15 (s, 3H, CH₃CO), 2.09 (s, 3H, CH₃CO), 2.04 (s, 3H, CH₃CO), 1.88 (s,3H, CH₃CO), 0.99 (s, 18H, 2×Boc). ¹³C NMR (75 MHz, CDCl₃): δ 171.2,171.1, 170.2, 169.8, 165.1 (d, J_(C2,F2) 32.8 Hz, C-1), 162.7, 157.5,152.9, 105.7 (dd, J_(C2,F2) 226.7 Hz, J_(C2,F3) 27.8 Hz, C-2), 90.4 (dd,J_(C3,F3) 191.8 Hz, J_(C3,F2) 31.9 Hz, C-3), 84.4, 80.3, 74.7, 69.3,67.2, 62.5, 53.7, 52.8 (dd, J_(C4,F3) 20.3 Hz, J_(C4,F2) 6.2 Hz, C-4),49.0 (d, J_(C5,F3) 5.0 Hz, C-5), 28.3 (3C), 28.1 93C), 23.1, 21.0 (2C),20.9. ¹⁹F NMR (282 MHz, CDCl₃): δ −115.6 (d, J_(F2,F3) 11.3 Hz, F-2 eq),−195.8 (d, F-3 eq). ESI-MS: 733.4 [(M+Na)⁺].

5-Acetamido-2,4,5-trideoxy-2α,3β-difluoro-4-guanidino-α-D-erythro-L-gluco-nonulopyranosonate(5)“5-Acetamido-3,4,5-trideoxy-2α,3β-difluoro-4-guanidino-α-D-erythro-L-gluco-nonulopyranosonate”:To a solution of 4 (71 mg, 0.1 mmol) in dry methanol (6 mL) was addedsodium methylate solution (5.4 M, 0.1 mL) under N₂, and the reactionmixture was stirred overnight at room temperature. The reaction wasneutralized with Amberlite (IR-120), filtered and washed with methanol,evaporated to give a residue. The resulting residue was dissolved intoTFA (1 mL) and stirred for 2 h at room temperature, evaporated andco-evaporated with toluene three times. The crude product was purifiedby flash column chromatography (EtOAc/MeOH/H₂O=7/2/1) to give compound 5as a white solid (26 mg, 92%). ¹H NMR (400 MHz, D₂O): δ 4.71 (ddd, 1H,J_(3,4) 8.9 Hz, J_(H3,F3) 48.8 Hz, J_(H3,F2) 13.4 Hz, H-3), 4.56 (ddd,1H, J_(H4,F3) 19.0 Hz, H-4), 4.49 (brd, 1H, H-6), 4.36 (t, 1H,J_(4,5)=J_(5,6) 10.5 Hz, H-5), 3.84 (dd, 1H, J_(8,9a) 2.6 Hz, H-9a),3.79 (m, 1H, H-8), 3.62 (dd, 1H, J_(8,9b) 6.0 Hz, J_(9a,9b) 11.5 Hz,H-9b), 3.56 (brd, 1H, J_(7,8) 9.1 Hz, H-7). ¹³C NMR (75 MHz, D₂O): δ174.6, 169.6 (d, J_(C2,F2) 30.8 Hz, C-1), 157.6, 106.8 (dd, J_(C2,F2)222.1 Hz, J_(C2,F3) 27.8 Hz, C-2), 91.5 (dd, J_(C3,F3) 188.1 Hz,J_(C3,F2) 31.5 Hz, C-3), 73.5, 69.9, 67.9, 63.2, 55.7 (dd, J_(C4,F3)18.8 Hz, J_(C4,F2) 8.2 Hz, C-4), 48.4 (d, J_(C5,F3) 6.4 Hz, C-5), 21.9.¹⁹F NMR (282 MHz, D₂O): δ −112.7 (d, J_(F2,F3) 12.7 Hz, F-2 eq), −199.2(d, F-3 eq). ESI-MS: 369.4 [(M-H)⁻].

5-Acetamido-2,4,5-trideoxy-4-azido-2α,3β-difluoro-α-D-erythro-L-gluco-nonulo-pyranosonate(6)“5-Acetamido-3,4,5-trideoxy-4-azido-2α,3β-difluoro-α-D-erythro-L-glucononulo-pyranosonate”:To a solution of 3 (50 mg, 0.1 mmol) in dry methanol (5 mL) was addedsodium methylate solution (5.4 M, 50 μL) under N₂, and the reactionmixture was stirred for 2 h at room temperature. To the reaction mixturewas added a couple drops of water, and stirred for another an hour atroom temperature. The reaction was neutralized with Amberlite (IR-120),filtered and washed with methanol, and evaporated to give a residue. Theresulting residue was purified by flash column chromatography(EtOAc/MeOH/H₂O=12/2/1) to give product 6 as a white solid (34 mg, 96%).¹H NMR (400 MHz, CD₃OD): δ 4.69 (ddd, 1H, J_(H4,F3) 19.7 Hz, H-4), 4.43(ddd, 1H, J_(3,4) 9.0 Hz, J_(H3,F3) 50.0 Hz, J_(H3,F2) 13.5 Hz, H-3),4.39 (brd, 1H, H-6), 4.11 (t, 1H, J_(4,5)=J_(5,6) 10.6 Hz, H-5), 3.79(dd, 1H, J_(8,9a) 2.8 Hz, H-9a), 3.77 (m, 1H, H-8), 3.64 (dd, 1H,J_(8,9b) 5.2 Hz, J_(9a,9b) 11.3 Hz, H-9b), 3.49 (brd, 1H, J_(7,8) 9.1Hz, H-7). ¹³C NMR (75 MHz, CD₃OD): δ 173.2, 169.0 (d, J_(C2,F2) 45.4 Hz,C-1), 106.5 (dd, J_(C2,F2) 222.6 Hz, J_(C2,F3) 28.0 Hz, C-2), 92.6 (dd,J_(C3,F3) 188.8 Hz, J_(C3,F2) 30.3 Hz, C-3), 73.9, 70.4, 68.4, 63.4,63.1 (dd, J_(C4,F3) 24.8 Hz, J_(C4,F2) 8.0 Hz, C-4), 49.1 (d, J_(C5,F3)6.3 Hz, C-5), 21.4. ¹⁹F NMR (282 MHz, CD₃OD): δ −115.7 (d, J_(F2,F3)11.3 Hz, F-2 eq), −199.6 (d, F-3 eq). ESI-MS: 353.2 [(M-H)⁻].

5-Acetamido-2,4,5-trideoxy-4-amino-2α,3β-difluoro-α-D-erythro-L-glucononulo-pyranosonate(7)“5-Acetamido-3,4,5-trideoxy-4-amino-2α,3β-difluoro-α-D-erythro-L-glucononulo-pyranosonate”A suspension of 6 (39 mg, 0.11 mmol) and Pd/C (10%, 12 mg) in drymethanol (8 mL) was vacuumed and filled with hydrogen for three times,and stirred overnight under H₂ atmosphere at room temperature. Thereaction mixture was filtered through a short pad of Celite and washedwith methanol. The organic solvent was evaporated to give a solid. Thesolid was dissolved in distilled water and filtered with MILLEX-GPfilter unit (pore size: 0.22 μm), and then lyophilized to give compound7 as a white solid (36 mg, 100%). ¹H NMR (400 MHz, D₂O): δ 4.83 (ddd,1H, J_(3,4) 9.1 Hz, J_(H3,F3) 49.6 Hz, J_(H3,F2) 13.2 Hz, H-3),4.46˜4.28 (m, 3H, H-4, H-5 & H-6), 3.84 (dd, 1H, J_(8,9a) 2.5 Hz, H-9a),3.79 (m, 1H, H-8), 3.62 (dd, 1H, J_(8,9b) 6.0 Hz, J_(9a,9b) 11.7 Hz,H-9b), 3.54 (brd, 1H, J_(7,8) 9.0 Hz, H-7). ¹³C NMR (100 MHz, D₂O): δ175.1, 169.2 (d, J_(C2,F2) 30.0 Hz, C-1), 106.4 (dd, J_(C2,F2) 222.0 Hz,J_(C2,F3) 28.0 Hz, C-2), 90.1 (dd, J_(C3,F3) 186.0 Hz, J_(C3,F2) 33.0Hz, C-3), 73.6, 69.9, 67.7, 63.2, 54.0 (dd, J_(C4,F3) 18.0 Hz, J_(C4,F2)7.0 Hz, C-4), 46.9 (d, J_(c5,F3) 6.0 Hz, C-5), 22.2. ¹⁹F NMR (282 MHz,D₂O): δ −113.6 (d, J_(F2,F3) 14.1 Hz, F-2 eq), −199.9 (d, F-3 eq).ESI-MS: 327.3 [(M-H)⁻].

5-Acetamido-5-deoxy-3-fluoro-D-erythro-β-L-manno-2-nonulopyranosonicacid fluoride (DFSA)

DFSA was synthesized according to the procedure of Watts and Withers (A.G. Watts, S. G. Withers, Can. J. Chem. 82, 1581 (2004)).

2-Keto-3-deoxy-3-fluoro-D-glycero-β-L-manno-2-nonulosonic acid fluoride(difluoroKDN)

DifluoroKDN was synthesized according to the procedure of Watts et al.(A. G. Watts et al., J. Biol. Chem. 281, 4149 (2006)).

Enzyme Kinetics Measurement of Inactivation and Reactivation KineticParameters Influenza A Neuraminidase

All experiments were carried out in a 20 mM Tris/50 mM CaCl₂ buffer, pH7.6. Cuvettes had a path length of 1 cm and were used in a Cary 4000UV/visible spectrophotometer connected to a circulating water bath. Thedata were analyzed using the program GraFit 4.0 (Erithacus software) (R.Leatherbarrow, Erithacus Software Ltd., 4th Edition, Staines, UK,(1990)). The viral stock solution was prepared by adding 300 μL ofbuffer, 50 μL 1% BSA and 50 μL 4% Triton X-100 to 100 μL of virussolution that had been treated with NP-40 to kill any viral infectivity.Time-dependent inactivations were performed by pre-incubating the viralstock (60 μL) at 30° C. in the presence of several concentrations ofinactivator (ranging from 0.05 μM to 10 μM), buffer and 1% BSA (20 μL),in a total volume of 200 μL. Residual enzyme activity was determined atappropriate time intervals by the addition of an aliquot of theinactivation mixture (30 μL) to an assay solution containing 0.75 mM4-trifluoromethylumbelliferyl sialic acid (CF₃MUSA). Kinetic parameterswere determined by measuring the initial linear increase in absorbanceat 400 nm. The initial rates at each time point were plotted as afunction of time to obtain time-dependent exponential decay curves fromwhich k_(i obs) could be obtained for each inactivator concentrationusing the equation:

(rate)_(t)=(rate)_(t=0) e ^((ki obs t))+offset

The inactivation rate constant (k_(i)) and the reversible dissociationconstant for the inactivator (K_(i)) were determined by fitting data fork_(i obs) versus inactivator concentration to the equation:

k _(i obs) =k _(i) [I]/(K _(i) +[I])

In the case where [I]<<K_(i), a second-order rate constant (k_(i)/K_(i))was determined by fitting the data to the equation:

k _(i obs) =k _(i) [I]/K _(i)

Time-dependent reactivation parameters were determined as follows.Inactivated enzyme solution (200 μl) was applied to a Ultrafree® 0.5Centrifugal Device 50 K filter (Millipore™) at 4° C. to remove excessinactivator. The filter was washed once with 200 μL buffer at 4° C. Theeluted enzyme was dissolved with 200 μL buffer. From this solution a 180μL aliquot was added to a solution of 1% BSA (60 μL) and buffer (360 μL)and incubated at 30° C. Enzyme activity was assayed at time intervals bythe addition of an aliquot of eluted enzyme (30 μL) to an assay solutioncontaining 0.75 mM CF₃MUSA, at 30° C. First-order rate constants forreactivation (k_(r hyd)) were determined by direct fit of the activityversus time data to a first-order equation.

Human Neu2

All experiments were carried out in a 100 mM Na₂HPO₄/50 mM Citric acidbuffer, pH 5.6. Cuvettes had a path length of 1 cm and were used in aCary 4000 UV/visible spectrophotometer connected to a circulating waterbath. The data were analyzed using the program GraFit 4.0 (Erithacussoftware) (R. Leatherbarrow, Erithacus Software Ltd., 4th Edition,Staines, UK, (1990)). Time-dependent inactivation studies were performedby pre-incubating the enzyme (60 μL) at 27° C. in the presence of 1 mMinactivators (FaxGuDFSA, FeqAmDFSA and FeqGuDFSA), buffer and 0.5% BSA(20 μL), in a total volume of 200 μL. Residual enzyme activity wasdetermined at appropriate time intervals by the addition of an aliquotof the inactivation mixture to an assay buffer solution containing 8 mM4-trifluoromethylumbelliferyl sialic acid (CF₃MUSA). The initial linearincrease in absorbance at 400 nm was used as a measure of the enzymeactivity. These initial rates at each time point were plotted as afunction of time to obtain time-dependent exponential decay curves fromwhich k_(i obs) could be obtained using the equation:

(rate)_(t)=(rate)_(t=0) e ^((ki obs t))+offset

IC₅₀ Enzyme Inhibition Assays

Zanamivir was obtained from GlaxoSmithKline (Stevenage, UK) andoseltamivir carboxylate was obtained from oseltamivir phosphate by Dr.Keith Watson (Walter and Eliza Hall Institute, Australia). Serialten-fold dilutions of inhibitors were prepared in water. The fluorescentsubstrate 4-methylumbelliferyl N-acetyl-α-D-neuraminic acid (MUNANA) waspurchased from Carbosynth (UK). Enzyme inhibition assays were carriedout as previously described (S. Barrett et al., PLoS One 6, e23627(2011); J. L. McKimm-Breschkin et al., J. Antimicrob. Chemother. 67,1874 (2012)) using a 30 min preincubation with virus and inhibitor, thenfluorescence was read after incubation with MUNANA for 60 min. The IC₅₀was calculated as the inhibitor concentration resulting in a 50%reduction in fluorescent units (FU) compared to the control.

Identification of the Active Site Nucleophile of Influenza NA Labelingand Proteolysis of NA N9

Labeling of N9 NA (1 mg/mL) was accomplished by incubating the enzyme(40 μL) in 50 mM phosphate buffer (pH 6.8), containing 2 mM 2,3-difluoroKDN (30 μL) for 30 min at room temperature. After this time, phosphatebuffer (120 μL, pH 2.0) containing pepsin (0.3 mg/mL) was added.Proteolytic digestion was performed for 1 h and the sample was frozenprior to mass spectrometric analysis. A sample of unlabeled enzyme forcomparison was also prepared in the same manner.

Electrospray Mass Spectrometry

Mass spectra were recorded on a PE-Sciex API 300 triple quadrupole massspectrometer and a PE-Sciex API QSTAR pulsar (Sciex, Thornhill, Ontario,Canada) equipped with an Ionspray ion source. Peptides were separated byreverse phase HPLC on a LC Packing UltiMate Micro HPLC system (Dionex,Sunnyvale, Calif.) directly interfaced with the mass spectrometer. Ineach of the MS experiments, the proteolytic digest was loaded onto aC-18 column (LC Packing, 100 Å pepMap, 1 mm×150 mm) equilibrated withsolvent A (solvent A: 0.05% trifluoroacetic acid-2% acetonitrile inwater). Elution of the peptides was accomplished using a gradient(0%-60%) of solvent B over 60 min followed by 85% solvent B over 20 min(solvent B: 0.045% trifluoroacetic acid-80% acetonitrile in water).Solvents were pumped at a constant flow rate of 50 μL/min. Spectra wererecorded in the single quadrupole scan mode (LC-MS) or the tandem MSproduct-ion scan mode. In the single quadrupole mode (LC-MS), thequadrupole mass analyzer was scanned over a mass-to-charge ratio (m/z)range of 100-2200 Da with a step size of 0.5 Da and a dwell time of 1.5ms per step. The ion source voltage (ISV) was set at 5.5 kV and theorifice energy (OR) was 45 V. In the tandem MS daughter-ion scan mode,the spectra were obtained in a separate experiment by selectivelyintroducing the labeled (m/z=1489) or unlabeled (m/z=1221) parent ionfrom the first quadrupole (Q1) into the collision cell (Q2) andobserving the product ions in the third quadrupole (Q3). The scan rangeof Q3 was 100-1600, the step size was 0.5 Da, the dwell time was 1 ms,ISV was 5 kV, OR was 45 V, Q0=−10, IQ2=−48.

X-Ray Crystallography

NA from influenza virus A/NWS/Tern/Australia/G70C/75 was purified asdescribed previously (T. J. Blick et al., Virology 214, 475 (1995)) andcrystallized in a similar fashion to that described (W. G. Laver et al.,Virology 137, 314 (1984)) in potassium phosphate buffer (1.7 M, pH 6.7).The NA-inhibitor N9-FeqGuDFSA complex was prepared by soaking crystalsin cryo-protectant solution containing well solution with 20% ofglycerol and 2 mM concentration of inhibitor over 35 min at 4° C. X-raydiffraction data was collected at −173° C. at the Australian synchrotronMX1 beamline (T. M. McPhillips et al., J. Synchrotron. Radiat. 9, 401(2002)) with wavelength of 0.95369 Å. The ADSC Q210 detector was set ata distance of 200 mm, 1° oscillations were taken and a total of 360frames were obtained, with each frame given a 1 s total exposure time.The data was processed with HKL2000 (Z. Otwinowski, W. Minor,Methods-Enzymol., 307 (1997)). A total of 2404581 (>1σ) observationswere measured up to 2.0 Å and reduced to 32402 unique reflections with amerging R_(merge)-factor of 16.6% over all the observations with mean<I>/<σ(I)> of 24.7. The space group is cubic, I432, with unit celldimension a=180.8 (2) Å. The position of the N9 molecule was identifiedin the asymmetric unit by PHASER (A. J. McCoy et al., J. Appl.Crystallogr. 40, 658 (2007)) molecular replacement using the structureof N9 without ligand (PDB entry: 1NNC) (J. N. Varghese, V. C. Epa, P. M.Colman, Protein Sci. 4, 1081 (1995)). The structure with N9 moleculesalone was refined and then the 3-fluoro(eq)-4-guanidino-sialyl moietywas built into the observed residual electron density. Initialrefinement of the inhibitor position did not account for the allresidual density observed, in particular in the region between theinhibitor and the Y406 residue. The continuous bridge of residualelectron density between the C-2 atom of the inhibitor and hydroxyloxygen OH of the aromatic side chain of Y406 residue suggested the C—Ocovalent bond (FIG. S4) Next, the two active site species, covalentlybonded and non-bonded elimination product were refined. The length ofthe covalent linkage for bonded species was kept at a chemicallysensible value (1.4 Å) by using corresponding stereochemical restraints.The occupancies of two species were refined to 30 and 70%, respectivelyfor bonded and non-bonded species, with sensible values of atomicB-factors. Additional water molecules in the active site and elsewherewere then identified by difference Fourier methods during the course ofthe refinement. Iterative refinement and model building were conductedusing REFMAC (G. N. Murshudov, A. A. Vagin, E. J. Dodson, Actacrystallogr. 53, 240 (1997)) and MIFit (D. E. McRee, J. Struct. Biol.125, 156 (1999); D. E. McRee, J. Badger, MIFit Manual© Rigaku,(2003-6)), and yielded a model for 388 residues for the N9 A chain with12 attached glycans, 7 glycerol molecules from cryo-protectant, 1calcium ion, two bound forms of the inhibitor (covalent and eliminationproduct) and 383 water molecules. The refinement was carried out usingthe full data set up to 2.0 Å with a 1 σ cutoff. The final R/R_(free)0.226/0.269 for the complex was 14.4/18.9% with rms deviations fromideal bonds and angles of 0.02 Å and 2.09°, respectively, and a meanB-value of 34.9 Å² for the refined non-hydrogen atoms. Progress of therefinement was monitored using the R_(free) statistics based on a testset encompassing 5% of the observed diffraction amplitudes (A. T.Brünger, Nature 355, 472 (1992)). The coordinates of the complex havebeen deposited in the PDB with deposition code 3W09 and furtherexperimental and data processing details are given in Table 3.

TABLE 3 X-ray data collection and refinement statistics. NA9-FeqGuDFSABeamline AS MX1 Wavelength (Å) 0.95369 Resolution range (Å) 42.66-2.0(2.05-2.00) Space group I432 Unit cell (a = b = c Å, α = β = γ = 90°)180.8 (2) Total reflections observed 2404581 Unique reflections 32402(2335) Redundancy 70 (35) Completeness (%) 99.55 (99.43) <I>/<σ(I)> 24.7(1.1) Wilson B-factor 53.40 R_(merge) ^(a) 0.166 (0.762) χ² _(merge)^(b) 1.37 (1.18) R^(c) 0.144 (0.267) R_(free) ^(d) 0.189 (0.294) Numberof atoms 3719 macromolecules 3245 ligands 48 water 383 cryo-agent 42metal 1 Protein residues 388 RMSD (bonds) (Å) 0.021 RMSD (angles) (°)2.092 Ramachandran favoured (%) 95.6 Ramachandran outliers (%) 0.5Average B-factor (Å²) 34.89 macromolecules 33.26 solvent 46.51 ligands30.85 Matthews coefficient, V_(m) (Å³/Da) 2.37 Solvent (%) 47.6Statistics for the highest-resolution shell are shown in parentheses.^(a)R_(merge) = Σ_(hkl)Σ_(j) |I_(j) − <I_(j)>|/Σ_(hkl)Σ_(j) | I_(j) |and ^(b)χ² _(merge) = Σ_(hkl)Σ_(j)(I_(j) − <I_(j)>)²/Σ_(hkl)Σ_(j)(σ_(j)² + <σ_(j)>²), where hkl specifies unique indices, j indicatesequivalent observations of hkl, I_(j) and σ_(j) ² are the observedintensities and their errors, and <Ij> and <σ_(j)> are the mean values.^(c)R = Σ_(hkl) || F_(o) | − F_(c) |/Σ_(hkl) | F_(o)|, where | F_(o) |and | F_(c) | are the observed and calculated structure factoramplitudes, respectively. ^(d)Represents 5% of the data.

Cell-Based Assay of Influenza Anti-Viral Activity

Madin Darby Canine Kidney (MDCK) cells were cultured as previouslydescribed (J. L. McKimm-Breschkin et al., J. Antimicrob. Chemother. 67,1874 (2012)). Plaque assays in MDCK cells were overlaid with DMEM/F12without serum using 0.5% immunodiffusion-grade agarose (MP Biomedicals,Australia), containing 1 mg/mL L-1-tosylamido-2-phenylethyl chloromethylketone (‘TPCK’)-treated trypsin (Worthington, USA). For plaque reductionassays (PRAs), serial ten-fold dilutions of inhibitors were incorporatedinto the overlay (J. L. McKimm-Breschkin et al., J Antimicrob.Chemother. 67, 1874 (2012)) which consists of making serial 2-folddilutions of the antiviral compounds (from 1:2 to 1:4096 in MegaVirmedium in enough volume for the number of viruses tested—60 uL pervirus), to which is added 100 infectious units of the specific influenzavirus and the preparations are transferred to monolayers of MDCK cellsin a microtitre plate. The assay was carried out on a 96-well microtitreplate. The plate is monitored for the development of influenzacytopathic effects from days 3 to 5 post infection, at which time theplate is fixed with 1% formalin, the agarose is removed and cells arestained with 0.05% neutral red and scanned. The IC₅₀ is the inhibitorconcentration causing a 50% decrease in plaque size. Where there wasgreater than 50% reduction in plaque size between two drugconcentrations a range is used. Antiviral activity is determined by theinhibition of development of cytopathic effects. The highest dilution ofthe compound at which the monolayers are intact is taken as theend-point. FaxGuDFSA, Zanamivir, Oseltamivir, and Peramivir were used ascontrols.

Dilution Preparations:

-   -   1. In row A on a clean 96-well microtitre plate, prepare 2-fold        serial dilutions of antiviral compounds from 1:2 to 1:4096 in        MegaVir medium in enough volume for the number of viruses tested        (60 uL per virus).    -   2. Transfer 55 uL of the 2-fold dilution series to a clean row        in the 96-well microtitre plate.    -   3. To the 55 uL dilution series, add 55 uL of diluted influenza        virus (at 100 TCID₅₀ per 25 ul). Also add virus to positive        control wells.    -   4. To the now 110 uL mixture, add 55 uL of 4× TPCK-treated        trypsin. Add trypsin also to positive and negative control        wells. Mix well.    -   5. Prepare also 2-fold serial dilutions from 1:2 to 1:256 for        the inoculating virus in MegaVir medium for back titration.

Plate Inoculation:

-   -   6. In a 96-well microtitre plate containing confluent monolayers        of MDCK cells in ˜200 uL MegaVir medium, transfer 75 uL of the        mixture to 2 respective rows as duplicates.    -   7. Transfer 50 uL of the positive control, and 25 uL of negative        controls to respective wells.    -   8. Transfer also 25 uL of the virus back titration in        duplicates.    -   9. Therefore in each well:        -   a. Samples: 25 uL compounds+25 uL virus+25 uL trypsin        -   b. Positive control: 25 uL virus+25 uL trypsin (no            compounds)        -   c. Negative control: 25 uL trypsin (no compounds or virus)        -   d. Back titration: 25 uL virus    -   10. The plates are incubated at 37° C. in a CO₂ incubator for 3        days, then observed for the appearance of cytopathic effects on        day 3 and day 5.

Viruses

The wild type and mutant viruses used in the plaque reduction assayand/or the MUNANA based enzyme inhibition assay were human strains:B/Perth/211/01 influenza B and D197E mutant (A. C. Hurt et al.,Antimicrob. Agents Chemother. 50, 1872 (2006)), with decreasedsusceptibility to all NA inhibitors due to E197 affecting interactionsof R152 with the N-acetyl group on the sugar ring (A. J. Oakley et al.,J. Med. Chem. 53, 6421 (2010)); A/Mississippi/3/01 H1N1 and H275Y mutant(A. S. Monto et al., Antimicrob. Agents Chemother. 50, 2395 (2006)) withdecreased susceptibility specifically to oseltamivir due to Y275limiting structural changes necessary to accommodate the oseltamivirpentyl ether side chain (P. J. Collins et al., Nature 453, 1258 (2008));A/Fukui/45/04 H3N2 and E119V mutant (M. Tashiro et al., Antivir. Ther.14, 751 (2009)), with decreased susceptibility specifically tooseltamivir due to altered interactions of V119 with the 4-amino groupon the cyclohexene ring. We also used the laboratory strain NWS/G70CH1N9, a reassortant containing the NWS HA and all other genes fromA/Tern/Australia/G70C/75 and the E119G mutant, as this mutant hasselective resistance to zanamivir, due to altered interactions of G119with the 4-guanidino group (T. J. Blick et al., Virology 214, 475(1995)). The NWS/G70C virus was also used as a source of purifiedprotein for mass spectroscopy, enzyme studies and crystallized for X-raycrystallography as previously described (T. J. Blick et al., Virology214, 475 (1995)). Virus was grown in eggs and the NA was proteolyticallycleaved using pronase and purified by gel filtration (T. J. Blick etal., Virology 214, 475 (1995)).

Some of the mutants were generated by producing viruses in derivativesof zanamivir, all of which still had the 4-guanidinium group. Several ofthe mutants have mutations at E119. E119 interactions are significantfor high affinity binding of NAIs, but each substitution often onlyaffects binding of a subset of the inhibitors. It is already known thatE119G confers zanamivir and peramivir resistance, but not oseltamivir,which is thought to be due to altered interactions with the guanidiniumgroup, and E119V confers oseltamivir and 4-aminoNeu5Ac2en resistance,but not to zanamivir or peramivir.

Wild Type Viruses:

*A/Auckland/3/2009 (pandemic H1N1)

*B/Florida/4/2006

*A/Solomon Islands/3/2006 (seasonal H1N1)

G70C H1N9 wt Fukui H3N2 wt

sH1N1/01sH1N1/08 wt

B/Perth wt Mutant Viruses:

*A/Auckland/3/2009 mutant 1 E119K*B/Florida/4/2006 mutant 1 E117D (E119D N2 numbering)*A/Solomon Islands/3/2006 mutant E119A

NWS/G70C H1N9 E119G Fukui H3N2 E119V

sH1N1/01 H275YsH1N1/08 H275Y

B/Perth D197E

*provided by Biota Holdings Limited

The H275Y numbering is based on the sequence of seasonal H1N1 strains,while the H274Y numbering is based on cross referencing and alignment tothe N2 strain. Generally, the H274Y numbering was how all numbering wasreferenced until the global spread of the H274Y H1N1 in 2007-8. However,there are several insertions and deletions between different subtypes,which made the numbering change. Accordingly, both are used and theyrefer to the same mutation (i.e. H275Y is the N1 numbering and the H274YN2 numbering). The H275Y mutants referred herein were identified H274Yin the provisional from which this application claims priority.

Animal Studies

Animal studies were performed in accordance with the recommendations inthe Guide to the Care and Use of Laboratory Animals of the CanadianCouncil on Animal Care. Ethics protocols were approved by the AnimalCare Committee of the University of British Columbia (A09-0058) and theUniversity Animal Care Committee of Simon Fraser University (956HS-10).

Pharmacokinetic Studies

Pharmacokinetics were determined in Balb/c mice (N=4 per group per timepoint). Compounds were administered in saline either intranasally orintravenously with a target dose of 1 mg/kg. At each time point, animalswere euthanized by CO₂ and blood was immediately drawn by cardiacpuncture, followed by organ collection. Plasma was separated from bloodbefore storage. All tissues were analyzed by extraction of the compound(either zanamivir or FaxGuDFSA) and analysis by UPLC-MS/MS methods. Thechromatography used a gradient mobile phase of A) 1% methanol inammonium acetate and B) acetonitrile and a HILIC stationary phasecolumn. Recovery of the analytes from tissue and limits of quantitation(based on accuracy (<25% bias) and precision (<20% RSD) werecharacterized for each method. FaxAmDFSA was used as an internalstandard. Extractions were accomplished with a mixture of ammoniumacetate and acetonitrile. For solid organs, samples were firsthomogenized with cycles in a BeadBeater apparatus followed bycentrifugation to remove solids from the sample. Data were analyzed todetermine pharmacokinetic parameters using WinNonLin 7.2.

Efficacy Study

Protective efficacy of FaxGuDFSA to influenza A virus challenge wasdetermined in 6-week old Balb/c mice. Mouse-adapted A/Hong Kong/1/68(H3N2) clone m20C (E. G. Brown et al., Proc. Natl. Acad. Sci. U.S.A. 98,6883 (2001)) was used as the challenge virus. Mice were challengedintranasally by 1,250 pfu (3×LD₅₀) virus in 10 μL DMEM. Mice weretreated with either FaxGuDFSA or zanamivir intranasally twice daily (in20 μL saline per dose) in the morning and the evening beginning 2 hprior to infection over 6 days. The intervals between treatments wereapproximately 8 and 16 h and thus, twelve doses in total wereadministered. Over the course of the experiment, mice were monitoredtwice daily for clinical signs and body weight. Animals were removedfrom the experiment when they lost 20% of body weight.

To monitor the virus replication in lung, virus RNA in the lung wasquantitated by qPCR amplification of the viral M genome segment andnormalization against the level of a house keeping gene, GAPDH. Lungswere harvested at predetermined time points and the RNA was extractedfrom the lungs (PureLink RNA Mini Kit, Ambion). The qPCR was performedwith probes that are labeled with 6-FAM or TAMRA, by using QuantiFastMultiplex RT-PCR Kit (Qiagen). Primer and probe sequences are availableupon request.

EXAMPLES

Further embodiments are described with reference to the following,non-limiting, examples.

Example 1 Difluorosialic Acids are Covalent NA Inhibitors that Reactwith Y406 as the Catalytic Nucleophile

NAs catalyze the hydrolysis of sialosides by a process resulting in netretention of stereochemistry at the site of substitution. As mentioned,a mechanism involving an ion-pair intermediate has long been suggestedfor the GH34 influenza NA (M. von Itzstein, Nat. Rev. Drug Discov. 6,967 (2007)). We herein provide the first evidence for a covalentintermediate formed in the course of the reaction catalyzed by theinfluenza NA by use of 3-fluorosialosyl fluoride (DFSA) (FIG. 1 a) as asubstrate that exhibits slow turnover. The electronegative fluorine atomat C-3 inductively destabilizes the oxocarbenium ion-like transitionstates for both formation and hydrolysis of the intermediate, thusslowing each step, while the C-2 anomeric fluoride leaving group speedsthe formation step, permitting accumulation of the covalent intermediate(FIG. 1 a). Rapid inactivation of NA was observed at low inactivatorconcentrations, such that individual kinetic parameters (K_(i) andk_(i)) could not be determined for the N9 NA; only a second order rateconstant k_(i)/K_(i) of 196 min⁻¹ mM⁻¹ could be measured (Table 4 andFIG. 2 a). Turnover of the covalent intermediate (k_(hydr)) alsooccurred rapidly, with a t_(1/2)<1 minute (Table 4 and FIG. 2 b).Confirmation of the formation of a covalent species and identificationof the site of attachment was achieved by peptic digestion of N9 NA thathad been labeled with 3-fluorosialyl fluoride (i.e. DFSA) or itsdifluoro KDN analogue (FIG. 3). Isolation and subsequent sequenceanalysis of the labeled peptide by LC/MS-MS identified this peptide asNTDWSGYSGSF, with the tyrosine (Y) bearing the sugar label. Thisprovides direct evidence for a role of Y406 as the catalyticnucleophile.

TABLE 4 Inactivation and reactivation parameters for DFSA. k_(i)/K_(i)t_(1/2 (reac)) Compound Virus (min⁻¹mM⁻¹) (min)

A/NWS/ G70C/75 196 <1 min

Previously published work by Hagiwara et al. (1994) reported3-fluoro-sialic acids as being only modest sialidase inhibitors.Specifically, they report two compounds, one with an OH group at carbon2 (position T in Formula I). However, the OH group is not a sufficientlygood leaving group to allow trapping of a covalent intermediate.Accordingly, the Hagiwara et al. OH compound (at C2 equivalent to T inFormula I) showed minimal inhibition. Furthermore, the other compoundtested by Hagiwara et al., which has a fluorine (a sufficient leavinggroup) at C2 (equivalent to T in Formula I), did not have the correctstereochemistry at C-2. Accordingly, an appreciation of theserequirements was missing in Hagiwara et al.

Example 2 Selective Inhibition of Influenza Virus NA In Vitro

With the knowledge that the influenza NAs employ a covalent mechanism,we embarked on a program to explore these 2,3-difluoro sialic acids(DFSAs) as a possible new class of mechanism-based influenzatherapeutics that inhibit by covalently blocking the active site. Thisis an attractive approach since not only can the initial affinity of thedrug (K_(i)) be optimized, but also, the relative rate constants forformation (k_(i)) and hydrolysis (k_(hydr)) of the trapped intermediate,with the objective being to optimize the ratio of k_(i)/k_(hydr). Such astrategy has worked well previously for the β-lactam antibiotics, andmay provide particularly favorable pharmacokinetic behavior in thissituation. Indeed covalent drugs are regaining respect, with three ofthe top-selling drugs in the U.S. being covalent inhibitors of theirtargets (J. Singh et al., Nature Rev. Drug Discov. 10, 307 (2011)). Keyimprovements required to convert DFSA into a useful drug candidate aretherefore to introduce selectivity for the viral NA over host enzymesand to drastically reduce rates of turnover (k_(hydr)). Since theincorporation of an equatorial cationic nitrogen substituent at the siteequivalent to OH-4 of sialic acid provided a substantial affinity boostwithin zanamivir and oseltamivir, it was of interest to synthesizeversions of DFSA bearing amine (Am) and guanidine (Gu) substituents atC-4. Not only might these electron-withdrawing substituents improve theinitial affinity and the specificity for the influenza enzyme, but alsoturnover of the intermediate may be further slowed, due both to theadditional stabilization of the bound intermediate provided throughimproved interactions with the enzyme, and to the added inductive effectof the substituent on the reaction transition state. The effects ofequatorial (eq) stereochemistry of F3 on inhibitory behavior were alsoexplored.

Synthesis of the protected diastereomeric3-equatorial-2,3-difluoro-4-azido neuraminic acids as key intermediateswas achieved by Selectfluor™ hydroxyfluorination of2,4-dideoxy-2,3-didehydro-4-azido-N-acetylneuraminic acid (4-azido-DANA)(M. von Itzstein et al., Carbohydr. Res. 244, 181 (1993)) followed byinstallation of an equatorial fluorine at C-2 using diethylaminosulfurtrifluoride (DAST). The lead candidates shown in Table 2, FeqAmDFSA andFeqGuDFSA, were then prepared by reduction or reductive guanidylation,followed by deprotection.

Kinetic Parameters for NA Inhibition

Kinetic parameters for inactivation and reactivation of N1, N2 and N9NAs, as representatives of the Group 1 and Group 2 enzymes, by severalDFSA derivatives are presented in Table 5. Rate constants for turnoverby hydrolysis (k_(hydr)) were determined by monitoring the time courseof reactivation of dialyzed, inactivated enzyme, and fitting the data toa first order expression. First order rate constants for inactivationand reactivation are also expressed in the form of half-lives for eachprocess in Table 5.

TABLE 5 Inactivation and reactivation parameters for DFSA derivatives.k_(i)/K_(i) K_(i) t_(1/2 (inac)) k_(hyd) t_(1/2 (reac)) CompoundVirus^(a) (min⁻¹mM⁻¹) (μM) (min) (min⁻¹) (min)

Brisbane H1N1 Brisbane H1N1 H275Y California H1N1 G70C H1N9 Hong KongH3N2  106  29    95  74  140 3.15 9.49   4.28 2.32 0.24 2.1 2.5   1.74.0 20.8  0.0001 0.0002   0.0001 0.0003 <0.0001   6900 3450   6900 2300>6900  

Brisbane H1N1 Brisbane H1N1 H275Y California H1N1 G70C H1N9 Hong KongH3N2  371  160    93  246  470 0.25 0.47   0.35 0.41 0.07 7.4 9.2  20.9  6.8 20.2  <0.0001   <0.0001     <0.0001   <0.0001  <0.0001   >6900   >6900     >6900   >6900   >6900  

Brisbane H1N1 Brisbane H1N1 H275Y California H1N1 G70C H1N9 Hong KongH3N2 3479  849   4422 4332 5662 0.23 5.50   0.37 0.21 — 0.9 0.2   0.40.8 — 0.0027 0.0059   0.0150 0.0140 0.0045  256  117    46  49  153

Brisbane H1N1 Brisbane H1N1 H275Y California H1N1 G70C H1N9 Hong KongH3N2 5812 2992   7594 3879 5737 0.35  0.5    0.1 0.35 0.15 0.3 0.5   0.90.5 0.8 0.0005 0.0015   0.001  0.0019 0.0005 1380  460    690  363 1380^(a)Brisbane H1N1 = A/Brisbane/59/07; Brisbane H1N1 H275Y =A/Brisbane/59/07 oseltamivir-resistant; California H1N1 =A/California/07/09; G70C H1N9 = A/NWS/G70C/75; Hong Kong H3N2 =A/HongKong/01/68; ^(b)N.D. =Not Determined.

Incorporation of the charged substituent at C-4 result in high initialaffinity and greatly reduces the rate constant for reactivation ofenzymes inactivated by the compounds in Table 5. Half-lives forreactivation of NAs labeled by these compounds ranged from 0.75 hto >100 h. These very slow turnover rates are extremely important asthey mean that the virus will remain inactivated for extended times,even after the compound may have been cleared from relevant tissues,with favorable consequences for pharmacokinetic behavior.

Interestingly, compounds with an equatorial fluorine at C-3 inactivatesignificantly faster than do those with an axial fluorine. Further, thepresence of a guanidine substituent at C-4 slows both the inactivationand reactivation more than does an amine substituent, with a muchgreater effect on the reactivation step. The half lives of 2-4 hours areexpected to be sufficient for 3F equatorial compounds to function wellin vivo. Since the two transition states (for formation and hydrolysis)will be very similar, this difference in rate likely has its origins inoptimized interactions of the guanidine with the active site at thestage of the covalent intermediate, as is seen in the crystal structureof the trapped species shown in FIG. 1 b. A covalent bond of 1.45 Å isclearly observed in the electron density map (not shown), between C-2 of3-fluoro (eq) sialic acid and the phenolic oxygen of Y406. As observedpreviously in a structure of the GH33 sialidase NanI (S. L. Newstead etal., J. Biol. Chem. 283, 9080 (2008)), the covalent intermediate speciesis accompanied by an unsaturated form of fluorosialic acid formed byelimination. The carboxylate groups of both bound species formelectrostatic interactions with the guanidine groups of the strictlyconserved arginine triad R118, R292 and R371, a key interaction commonamong existing NA inhibitors. However, the elimination product formsstronger interactions (≦3 Å) with R118 and R371 similar to those formedwith zanamivir, while the covalent intermediate exhibits shortercontacts (≦3 Å) with R292 (FIG. 1 b). In addition to many otherinteractions that have been observed in previous complexes of N9 NA withzanamivir (J. N. Varghese et al., Protein Sci. 4, 1081 (1995)), bothforms of FeqGuDFSA also form electrostatic interactions at 2.9 Å (FIG. 1b) between the equatorial fluorine and the positive charge of theguanidine group of R118, reminiscent of those seen in otherprotein/ligand systems (J. A. K. Howard et al., Tetrahedron 52, 12613(1996)), possibly explaining the higher initial affinity of theFeqGuDFSA inhibitor compared to others. The C-4 guanidine indeed formsstrong interactions with the anionic pocket, very similar to those foundwith zanamivir (M. von Itzstein et al., Nature 363, 418 (1993)), therebycontributing to stabilization of the intermediate.

Comparison of NA Inhibition

IC₅₀ values were measured for each of DFSA and its derivatives, as wellas zanamivir and oseltamivir carboxylate (oseltamivir from which thepro-drug ester had been hydrolyzed), against the NA of different virusstrains after pre-incubation for 30 minutes prior to substrate addition.The IC₅₀ values so obtained are presented in Tables 6-10, along withdata for mutant viruses to be discussed below.

TABLE 6 Effects of mutations at E119A, D, K on sensitivity to inhibitors(IC₅₀ nM) B/Florida Sol Isl sH1N1 Auckland pH1N1 nM wt E119D Fold Res wtE119A Fold Res wt E119K Fold Res Zanamivir 3.9 >10,000 ≧10,0002.2 >10,000 ≧10,000 0.6 >100,000 ≧100,000 Oseltamivir 22.6 1205 53 1.0419 419 0.2 147 613 FaxGuDFSA 6.6 1515 229 181 >10,000 >1000284 >100,000 >1000 FeqGuDFSA 2.0 27 13 4.7 130 27 4.0 >100,000 ≧10,000

The results shown in TABLE 6 are consistent with previous testing ofother panels of viruses with FaxGuDFSA. Notably, the FeqGuDFSA has alower IC₅₀ than the FaxGuDFSA for many of the resistant strains.

The E119K mutation appears to confer some resistance to both FaxGuDFSAand FeqGuDFSA, but less so to oseltamivir (4-NH₂), suggesting that theinteractions of the 4-guanidinium group are potentially significant tohigh affinity binding of not only the established NAIs but also to bothFaxGuDFSA and FeqGuDFSA. Thus the 3F in the equatorial position resultsin significantly different binding behaviour for the FeqGuDFSA comparedto the other inhibitors with a 4-G group.

For the E119D there is lower resistance with the FeqGuDFSA than theFaxGuDFSA, and importantly also several orders of magnitude lowerresistance than to zanamivir. Since the E119D is significantly lessresistant to FeqGuDFSA than to zanamivir, this suggests the guanidiniumin the fluorosialics is less likely to select for resistant strains thanthat in zanamivir, likely due to the different (transient covalent) modeof action.

TABLE 7 Effects of mutations at E119G, V on sensitivity to inhibitors(IC₅₀ nM) G70C G70C Fold Fukui Fukui Fold H1N9 H1N9 resis- H3N2 H3N2resis- wt E119G tance wt E119V tance Zanamivir 2.7 678 248 3.8 3.4 0.9Oseltamivir 2.8 2.9 1.1 1.7 260 155 FaxGuDFSA 66.7 1433 21 2006 998 0.5FeqGuDFSA 136 17.3 0.1 24.9 2.4 0.1

The E119G mutation appears to confer a high level resistance tozanamivir only, confers 20-fold resistance to FaxGuDFSA, whereas theFeqGuDFSA actually appears to bind better in the mutant than in the wildtype.

The E119V mutation appears to confer a high level resistance toOseltamivir, but does not confer resistance to the FaxGuDFSA, zanamivir,or FeqGuDFSA.

TABLE 8 Effects of H275Y mutation on sensitivity to inhibitors (IC₅₀ nM)Fold Fold sH1N1/01 sH1N/01 resis- sH1N1/08 sH1N1/08 resis- wt H275Ytance wt H275Y tance Zanamivir 1.9 2.2 1.2 1.0 2.0 2.0 Oseltamivir 3.12440 781 3.0 2000 667 FaxGuDFSA 115.4 217 1.9 136 265 1.9 FeqGuDFSA 12.643.5 3.5 6.8 43.2 6.4

FaxGuDFSA or FeqGuDFSA appear to still be effective against the H275Ymutation which confers high level oseltamivir resistance, but not tozanamivir.

TABLE 9 Effects of D197E mutation on sensitivity to inhibitors (nM)B/Perth wt B/Perth D197E Fold resistance Zanamivir 8.9 257.5 28.9Oseltamivir 104.4 708.0 6.8 FaxGuDFSA 54.0 161.9 3.0 FeqGuDFSA 4.5 8.01.8

Mutations at D197E appears to confer cross resistance to known NAIs dueto altered interactions with the adjacent R152 and the N-acetyl group onthe ring. However, the FeqGuDFSA does not appear to be affected by thisinteraction.

As shown in Table 10, comparison between pairs of DFSA derivatives againconfirms that, in almost all cases, each compound with an equatorialfluorine is a superior inhibitor to its epimer with an axial fluorine,indicating that, under these conditions, improved rates of inactivationare important. Likewise, in each case the guanidine derivative showedsuperior performance to its amine analogue. Consequently, FeqGuDFSA wasthe optimal derivative within the series. Comparison of these IC₅₀values with those for zanamivir and oseltamivir reveals that, on thismeasure, the compounds with an equatorial fluorine are of comparableefficacy, particularly when the guanidine is present. Further, their“on-rates” are superior to those of zanamivir, while off-rates areenormously slower (M. von Itzstein et al., Nature 363, 418 (1993); P. J.Collins et al., Nature 453, 1258 (2008); E. van der Vries et al., PLoSPathog. 9, (2012)).

TABLE 10 IC₅₀ values (nM) in the enzyme inhibition assay for wild typeand mutant pairs. FaxAm FaxGu FeqAm FeqGu Virus^(a) ZanamivirOseltamivir DFSA DFSA DFSA DFSA DFSA B/Perth 8.9 104.4 70 210 54 5.4 4.5B/Perth D197E 257.5 708.0 170 340 162 16 8 A/Mississippi H1N1 1.9 3.1 80840 115 45 13 A/Mississippi H1N1 2.2 2440 120 2210 217 86 44 H275YA/Fukui H3N2 3.8 1.7 1380 4710 2006 71 25 A/Fukui H3N2 3.4 260.0 2402440 998 265 2.4 E119V G70C H1N9 2.7 2.8 1190 2700 66.7 270 140 G70CH1N9 E119G 678.4 2.9 1150 1600 1433 73 17 The IC₅₀ is the concentrationof inhibitor which reduces enzyme activity by 50% compared to thecontrol uninhibited value. Values are the means of duplicate assays.^(a)B/Perth = B/Perth/211/01; B/Perth D197E = B/Perth/211/01oseltamivir-resistant; A/Mississippi H1N1 = A/Mississippi/3/01;A/Mississippi H1N1 H275Y = A/Mississippi/3/01 oseltamivir-resistant;A/Fukui H3N2 = A/Fukui/45/01; A/Fukui H3N2 E119V =A/Fukui/45/01oseltamivir-resistant; G70C H1N9 = A/NWS/G70C/75; G70C H1N9E119G = A/NWS/G70C/75 zanamivir-resistant.

Inhibitor Specificity

The specificity of these inhibitors was then evaluated by testing themagainst Neu2 as a representative human NA (all human NAs belong to thesequence-related family GH33). No inactivation of Neu2 was seen witheither of the amine derivatives, and inactivation by FaxGuDFSA andFeqGuDFSA occurred slowly, but at rates some 10⁵-10⁶ lower thaninactivation of NA at comparable concentrations. This behavior is aconsiderable improvement over zanamivir, which inhibits Neu2 with aK_(i) of 17 μM (22).

As seen above, the 3′ equatorial compounds are better at maintainingpotency against some viral strains than the 3′ axial counterparts.Furthermore, various resistant strains of virus appear to remainsensitive to the 3′ equatorial compounds even when sensitivitydiminishes for the 3′ axial compounds. In addition, the amine isgenerally expected to have better oral bioavailability than theguanidine.

Example 3 Effects on Influenza Virus Replication and Efficacy AgainstResistant Strains

Based upon these very promising results the ability of our DFSAderivatives to inhibit the replication of the virus in cell culture wasexplored using MDCK cells. Three A strains (N1, N2 and N9) plus one Bstrain were tested in plaque size reduction assays (PRA), with zanamivirused as a control. While absolute sensitivity in the PRA depends on boththe affinity of the HA for cell receptors as well as the NA function (J.L. McKimm-Breschkin, Antiviral Res. 47, 1 (2000); M. Tisdale, Rev. Med.Virol. 10, 45 (2000)), it is a useful assay for determining relativesensitivity to inhibitors. The PRA showed that all DFSA derivativesinhibited virus replication (Table 11) with no cytotoxicity observedeven at 5 mM concentrations of the derivatives. The H3N2 virus was lesssusceptible to all inhibitors compared to the other strains, due to thelower affinity of its HA, which allows viral spread with less NAactivity (C. I. Thompson et al., J. Antimicrob. Chemother. 53, 759(2004)). However, for all strains, substitution with the 4-aminoenhanced inhibition over the parent DFSA, and the 4-guanidinosubstitution further enhanced inhibition of virus replication. It isinteresting that the stereochemistry of the fluorine substituent hadlittle effect on efficacy for the compounds with a 4-amine substituent,apart from possibly a small enhancement with the B virus. However thepresence of an equatorial fluorine in the 4-guanidine version resultedin a further 10-fold enhancement in potency for three strains tested,the one exception being G70C (H1N9), which was particularly wellinhibited by the axial version also. Consequently, FeqGuDFSA had thehighest potency of all of the inhibitors, including zanamivir, againstthe influenza B virus and performed comparably against the influenza Astrains. Two points are particularly noteworthy at this stage. One isthat this behavior in PRA largely mirrors the in vitro kinetic data,with the compounds possessing an equatorial fluorine being superior tothose with an axial fluorine, and the 4-guanidine substitution beingsuperior to a 4-amino. The other is that the absolute IC₅₀ values inthese PRAs are consistently lower than those for enzyme inhibition forall except FeqAmDFSA and impressively in all cases are in the lownanomolar range. The relatively poor PRA data observed with FeqAmDFSAlikely reflects the faster reactivation of NA inactivated by thiscompound.

TABLE 11 IC₅₀ values in the plaque size reduction assay. FaxAm FaxGuFeqAm FeqGu Virus^(a) Zanamivir DFSA DFSA DFSA DFSA DFSA B/Perth 10 nM 1μM 100 nM 10-100 nM 10-100 nM  1 nM A/Mississippi H1N1 ≦1 nM 1 μM 100 nM10 nM 100 nM  1 nM A/Fukui H3N2 100 nM 100 μM 1 μM 100 nM 1 μM 10 nMG70C H1N9 1-10 nM 1-10 μM 1 μM 1-10 nM 1 μM 10 nM The IC₅₀ is theconcentration of inhibitor required to reduce plaque size by 50%. Valuesare the means of duplicate assays. ^(a)B/Perth = B/Perth/211/01;A/Mississippi H1N1 = A/Mississippi/3/01; A/Fukui H3N2 = A/Fukui/45/01;G70C H1N9 = A/NWS/G70C/75.

The DFSA derivatives proved effective in vitro against a series ofresistant strains, with the FeqGuDFSA again proving to be the mostwidely active (Tables 6-10). All compounds proved effective againstvirus with the H275Y mutation that affects binding of the isopentyl sidechain of oseltamivir, as might be predicted. This is also reflected inthe very similar kinetic data measured for each DFSA derivative withanother H275Y oseltamivir resistant strain and its parent (Tables 4 and5). While the E119G mutation, which affects interactions with theinhibitor's guanidine (J. N. Varghese et al., Structure 6, 735 (1998))led to a 20-fold reduction in efficacy of FaxGuDFSA, this was much lesssevere than the 250-fold reduction in zanamivir binding. Theeffectiveness of the FeqGuDFSA is particularly noteworthy, andhighlights the different resistance profiles and modes of action of theDFSAs and zanamivir. Clearly, selection against transition stateanalogue binding (zanamivir) does not suppress covalent intermediateformation. Indeed in the case of the E119 mutations that target the4-position, FeqGuDFSA actually performed 10-fold better against themutant strains than against the wild type. This most likely reflectsslower reactivation of the trapped intermediate formed on the mutant,due to disruption of transition state-stabilizing interactions. Thisexcellent profile against otherwise resistant strains is extremelypromising and supports the concept of mechanism-based inhibition as ameans to minimize selection of resistant strains.

Example 4 In Vivo Efficacy Studies

Prior to in vivo efficacy studies in mouse models, the pharmacokineticproperties of FaxGuDFSA as a representative DFSA derivative wereevaluated for administration by intravenous and intranasal routes, andcompared with data collected in parallel for zanamivir. Levels ofFaxGuDFSA in blood, lung and trachea were measured and half livesdetermined. Intranasal dosing resulted in 92% bioavailability comparedto that seen with intravenous injection, along with approximately seventimes higher peak concentrations (Cmax) and ten times higher totalexposure (AUC_((0-120 min))) in both lung and trachea compared to theintravenous route. Further, the plasma half-life of FaxGuDFSA was alsosignificantly longer after intranasal administration than after IVinjection (Table 12). The general similarity of this pharmacokineticbehavior to that of zanamivir, consistent with the similar polarities ofthe two compounds, encouraged us to test the efficacy via intranasaladministration, using zanamivir as our control.

TABLE 12 Pharmacokinetic data on distribution of analytes afterintranasal delivery. Analyte Assay parameter Plasma Lung TracheaZanamivir Recovery  31% 55% Not evaluated LOQ^(a) 5 ng/mL 20 ng/g 500ng/g Gradient (A > B) 85 > 45% 80 > 40% FaxGuDFSA Recovery 102% 74% 69%LOQ^(a) 5 ng/mL  4 ng/g  50 ng/g Gradient (A > B) 98 > 30% ^(a)LOQ =Limit of quantitation.

Efficacy tests were conducted using a mouse-adapted influenza A virusstrain, A/Hong Kong/1/68 (H3N2) (E. G. Brown et al., Proc. Natl. Acad.Sci. U.S.A. 98, 6883 (2001)). Balb/c mice were treated with either DFSAderivative, zanamivir or saline twice daily by intranasaladministration, starting two hours prior to infection. Body weights andgeneral condition were monitored and when the animals lost 20% bodyweight they were euthanized and scored as non-survivors. In an initialstudy using 1 mg/kg/day of DFSA derivatives FaxGuDFSA showed superiorresults to FaxAmDFSA in prolonging the survival of animals. WhenFaxGuDFSA was tested at a higher dose of 10 mg/kg/day it protected allthe mice from the lethal infection, as did zanamivir (FIG. 4 a). Inorder to confirm that the compounds were acting as anti-viral agents,the viral RNA loads in lung tissue were measured by qPCR. These resultsconfirmed that survival was indeed associated with suppression of viralreplication, in a similar manner to the effect of zanamivir.

Dose-dependency of FeqGuDFSA in the protection of mice was demonstratedwith 100% efficacy at 10 mg/kg/day and less protection at lower doses(FIG. 4 b and Table 13). The compounds induced no ill-effect in thetreated animals during the experiments compared to the saline control.

TABLE 13 Efficacy of DFSAs with H3N2 influenza-infected mice. Delay intime to endpoint Survival Dose relative to untreated Frequency Treatment(mg/kg/d) infected control^(‡) (N, %) Evaluation of FaxGuDFSA at 10mg/kg/d dose, N = 10 per group Untreated Infected 0 1     0 controlZanamivir 10 N/A 100^(†) FaxGuDFSA 10 N/A 100^(†) Evaluation ofFeqGuDFSA at 1, 3 & 10 mg/kg/d dose, N = 5 per group Untreated Infected0 1     0 Control Untreated Uninfected 0 N/A 100^(†) Control Zanamivir 11.2*   0 3 1.7***  0 FeqGuDFSA 1 1.2    0 3 1.5***   20^(††) 10 N/A100^(†) ^(‡)Delay in time to endpoint is expressed in terms of foldchange relative to the untreated infected control group in eachexperiment (1, 2 or 3). When all animals survived and did not reach theendpoint, no delay was calculated, denoted with “N/A”. In experiment #1the humane endpoint was established at 15% body weight loss whereas inexperiments #2 and 3, the endpoint was 20% body weight loss.*Statistically significant delay in mean time to reach the endpoint(1-way ANOVA p < 0.05). ***Statistically significant delay in mean timeto reach the endpoint (1-way ANOVA p < 0.001). ^(†)All groups treatedwith a 10 mg/kg/d dose of NAI were 100% protected from reaching thehumane endpoint, which was statistically significant (Mantel-Cox p <0.0001). ^(††)FeqGuDFSA at 3 mg/kg/d gave 20% protection from reachingthe endpoint, which was statistically significant (Mantel-Cox p <0.004).

CONCLUSIONS

The enzyme IC₅₀ kinetics, reactivation, plaque reduction assays andcross-resistance data all suggest the FeqGuDFSA is a superior inhibitorto the FaxGuDFSA. Resistance data also supports the FeqGuDFSA as havinga different resistance profile than zanamivir, oseltamivir, andFaxGuDFSA. Thus the 3′ equatorial F leads to novel interactions of the4-G group at the ground and transition states, thus avoiding resistanceseen with many mutations at E119. Fluorinated compounds of the classdescribed herein are inhibitors of a range of glycosidases, and specificwith respect to their target enzymes. These compounds aremechanism-based in their inhibitory action. They bind to the enzyme likethe normal substrate and undergo the first step of catalysis(intermediate formation) like the natural substrate, but then only veryslowly undergo the second step (turnover via hydrolysis). Importantly,this mechanism-basis inhibition should make resistance formation byviruses more difficult. Since the inhibitors are mechanism-based, anymutations in the viral enzyme that reduce the inhibition mustnecessarily reduce the efficiency of the enzyme on the naturalsubstrate. Please note that the sialic acid numbering is different fromthat of aldose-sugars due to the anomeric carboxylate.

The fluorosialics described herein differ fundamentally from zanamivirand oseltamivir in two major ways. Zanamivir and oseltamivir arereversibly binding inhibitors that interact with the enzyme active sitevery tightly due to their flattened, cyclic conformation. Their bindingmode likely imitates the transition state conformation of the sugarduring hydrolysis. They are therefore transition state mimics. Thefluorosialics described herein, by contrast, contain no double bond thusadopt a regular chair conformation. They react with the enzyme as ifthey are substrates and form a covalent bond with the active sitenucleophile, and only hydrolyse to products very slowly. They derivetheir very high efficacies primarily from the long-lived nature of theintermediate formed.

It was not evident that a 3′ equatorial F substituent would increase theeffectiveness of these compounds against resistant viral strains strainsas compared to stereoisomers having 3′ axial F configuration.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. The word “comprising” isused herein as an open-ended term, substantially equivalent to thephrase “including, but not limited to”, and the word “comprises” has acorresponding meaning. As used herein, the singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a thing” includes more thanone such thing. Citation of references herein is not an admission thatsuch references are prior art to the present invention.

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What is claimed is:
 1. A compound of formula I:

wherein T is COOH or COOR¹, wherein R¹ is a C₁₋₂₀ linear, branched orcyclic, saturated or unsaturated, unsubstituted alkyl group, Z is F, orCl; D is F, or Cl; X is NH₂, NHC(NH)NH₂, NHCH₃, NHCH₂CH₃, NHCH₂CH₂CH₃,NHCH₂CH₂CH₂CH₃, or NHC(CH₃)CH₃; Q is OH, OMe, or OAc; E is OH, or OAc;and A is OH, or OAc.
 2. The compound of claim 1, wherein R¹ is a C₁₋₁₀.3. The compound of claim 1 or 2, wherein T is COOEt; Z is F; D is F; Xis NH₂ or NHC(NH)NH₂; Q is OH; E is OH; and A is OH.
 4. The compound ofclaim 1, wherein T is COOH; Z is F; D is F; X is NH₂ or NHC(NH)NH₂; Q isOH; E is OH; and A is OH.
 5. The compound of claim 1 or 4, wherein T isCOOH; Z is F; D is F; X is NH₂; Q is OH; E is OH; and A is OH.
 6. Thecompound of claim 1 or 4, wherein T is COOH; Z is F; D is F; X isNHC(NH)NH₂; Q is OH; E is OH; and A is OH.
 7. A compound having theformula:


8. A compound having the formula:


9. A method of treating a viral infection comprising administering to asubject in need thereof an effective amount of the compound of any oneof claims 1 to 8 or an effective amount of a pharmaceutically acceptablesalt thereof.
 10. The method of claim 9 wherein the compound, orpharmaceutically acceptable salt thereof, inhibits viral neuraminidase.11. The method of claim 9 or claim 10, wherein the viral infection iscaused, at least in part, by an influenza virus.
 12. The method of claim11, wherein the influenza virus is an H1N1, H3N2 or H1N9 subtype. 13.The method of claim 11, wherein the influenza virus is resistant tozanamivir.
 14. The method of claim 11, wherein the influenza virus isresistant to oseltamivir.
 15. The method of claim 11, wherein theinfluenza virus is resistant to peramivir.
 16. A pharmaceuticalcomposition comprising the compound of any one of claims 1 to 8 and apharmaceutically acceptable excipient or carrier.
 17. Use of a compoundof any one of claims 1 to 8 or an effective amount of a pharmaceuticallyacceptable salt thereof for the treatment of a viral infection.
 18. Useof a compound of any one of claims 1 to 8 or an effective amount of apharmaceutically acceptable salt thereof in the manufacture of amedicament for treating a viral infection.
 19. A compound of any one ofclaims 1 to 8 or an effective amount of a pharmaceutically acceptablesalt thereof for the treatment of a viral infection.